Surgical irrigation solution and method for inhibition of pain and inflammation

ABSTRACT

A method and solution for perioperatively inhibiting a variety of pain and inflammation processes at wounds from general surgical procedures including oral/dental procedures. The solution preferably includes multiple pain and inflammation inhibitory at dilute concentration in a physiologic carrier, such as saline or lactated Ringer&#39;s solution. The solution is applied by continuous irrigation of a wound during a surgical procedure for preemptive inhibition of pain and while avoiding undesirable side effects associated with oral, intramuscular, subcutaneous or intravenous application of larger doses of the agents. One preferred solution to inhibit pain and inflammation includes a serotonin 2  antagonist, a serotonin 3  antagonist, a histamine antagonist, a serotonin agonist, a cyclooxygenase inhibitor, a neurokinin 1  antagonist, a neurokinin 2  antagonist, a purinoceptor antagonist, an ATP-sensitive potassium channel opener, a calcium channel antagonist, a bradykinin 1  antagonist, a bradykinin 2  antagonist and a μ-opioid agonist.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of prior application Ser. No.09/109,885, filed Jul. 2, 1998, which is a continuation of priorapplication Ser. No. 08/670,699, filed Jun. 26, 1996, now U.S. Pat. No.5,820,583, which is a continuation-in-part of prior Internationalapplication serial number PCT/US95/16,028, filed Dec. 12, 1995, anddesignating the U.S., which is a continuation-in-part of priorapplication Ser. No. 08/353,775, filed Dec. 12, 1994, now abandoned,priority from the filing dates of which is hereby claimed under 35U.S.C. §120.

I. FIELD OF THE INVENTION

The present invention relates to surgical irrigation solutions andmethods, and particularly for anti-inflammatory, anti-pain, anti-spasmand anti-restenosis surgical irrigation solutions.

II. BACKGROUND OF THE INVENTION

Arthroscopy is a surgical procedure in which a camera, attached to aremote light source and video monitor, is inserted into an anatomicjoint (e.g., knee, shoulder, etc.) through a small portal incision inthe overlying skin and joint capsule. Through similar portal incisions,surgical instruments may be placed in the joint, their use guided byarthroscopic visualization. As arthroscopists' skills have improved, anincreasing number of operative procedures, once performed by “open”surgical technique, now can be accomplished arthroscopically. Suchprocedures include, for example, partial meniscectomies and ligamentreconstructions in the knee, shoulder acromioplasties and rotator cuffdebridements and elbow synovectomies. As a result of widening surgicalindications and the development of small diameter arthroscopes, wristand ankle arthroscopies also have become routine.

Throughout each arthroscopy, physiologic irrigation fluid (e.g., normalsaline or lactated Ringer's) is flushed continuously through the joint,distending the joint capsule and removing operative debris, therebyproviding clearer intra-articular visualization. U.S. Pat. No 4,504,493to Marshall discloses an isomolar solution of glycerol in water for anon-conductive and optically clear irrigation solution for arthroscopy.

Irrigation is also used in other procedures, such as cardiovascular andgeneral vascular diagnostic and therapeutic procedures, urologicprocedures and the treatment of burns and any operative wounds. In eachcase, a physiologic fluid is used to irrigate a wound or body cavity orpassage. Conventional physiologic irrigation fluids do not provideanalgesic, anti-inflammatory, anti-spasm and anti-restenotic effects.

Alleviating pain and suffering in postoperative patients is an area ofspecial focus in clinical medicine, especially with the growing numberof out-patient operations performed each year. The most widely usedagents, cyclooxygenase inhibitors (e.g., ibuprofen) and opioids (e.g.,morphine, fentanyl), have significant side effects includinggastrointestinal irritation/bleeding and respiratory depression. Thehigh incidence of nausea and vomiting related to opioids is especiallyproblematic in the postoperative period. Therapeutic agents aimed attreating postoperative pain while avoiding detrimental side effects arenot easily developed because the molecular targets for these agents aredistributed widely throughout the body and mediate diverse physiologicalactions. Despite the significant clinical need to inhibit pain andinflammation, as well as vasospasm, smooth muscle spasm and restenosis,methods for the delivery of inhibitors of pain, inflammation, spasm andrestenosis at effective dosages while minimizing adverse systemic sideeffects have not been developed. As an example, conventional (i.e.,intravenous, oral, subcutaneous or intramuscular) methods ofadministration of opiates in therapeutic doses frequently is associatedwith significant adverse side effects, including severe respiratorydepression, changes in mood, mental clouding, profound nausea andvomiting.

Prior studies have demonstrated the ability of endogenous agents, suchas serotonin (5-hydroxytryptamine, sometimes referred to herein as“5-HT”), bradykinin and histamine, to produce pain and inflammation.Sicuteri, F., et. al., Serotonin-Bradykinin Potentiation in the PainReceptors in Man, Life Sci. 4, pp. 309-316 (1965); Rosenthal, S. R.,Histamine as the Chemical Mediator for Cutaneous Pain, J. Invest.Dermat. 69, pp. 98-105 (1977); Richardson, B. P., et. al.,Identification of Serotonin M-Receptor Subtypes and their SpecificBlockade by a New Class of Drugs, Nature 316, pp. 126-131 (1985);Whalley, E. T., et. al., The Effect of Kinin Agonists and Antagonists,Naunyn-Schmiedeb Arch. Pharmacol. 36, pp. 652-57 (1987); Lang, E., et.al., Chemo-Sensitivity of Fine Afferents from Rat Skin In Vitro, J.Neurophysiol. 63, pp. 887-901 (1990).

For example, 5-HT applied to a human blister base (denuded skin) hasbeen demonstrated to cause pain that can be inhibited by 5-HT₃ receptorantagonists. Richardson et al., (1985). Similarly, peripherally-appliedbradykinin produces pain which can be blocked by bradykinin receptorantagonists. Sicuteri et al., 1965; Whalley et al., 1987; Dray, A., et.al., Bradykinin and Inflammatory Pain, Trends Neurosci. 16, pp. 99-104(1993). Peripherally-applied histamine produces vasodilation, itchingand pain which can be inhibited by histamine receptor antagonists.Rosenthal, 1977; Douglas, W. W., “Histamine and 5-Hydroxytryptamine(Serotonin) and their Antagonists”, in Goodman, L. S., et. al., ed., ThePharmacological Basis of Therapeutics, MacMillan Publishing Company, NewYork, pp. 605-638 (1985); Rumore, M. M., et. al., Analgesic Effects ofAntihistaminics, Life Sci 36, pp. 403-416 (1985). Combinations of thesethree agonists (5-HT, bradykinin and histamine) applied together havebeen demonstrated to display a synergistic pain-causing effect,producing a long-lasting and intense pain signal. Sicuteri et al., 1965;Richardson et al., 1985; Kessler, W., et. al., Excitation of CutaneousAfferent Nerve Endings In Vitro by a Combination of InflammatoryMediators and Conditioning Effect of Substance P, Exp. Brain Res. 91,pp. 467-476 (1992).

In the body, 5-HT is located in platelets and in central neurons,histamine is found in mast cells, and bradykinin is produced from alarger precursor molecule during tissue trauma, pH changes andtemperature changes. Because 5-HT can be released in large amounts fromplatelets at sites of tissue injury, producing plasma levels 20-foldgreater than resting levels (Ashton, J. H., et. al., Serotonin as aMediator of Cyclic Flow Variations in Stenosed Canine Coronary Arteries,Circulation 73, pp. 572-578 (1986)), it is possible that endogenous 5-HTplays a role in producing postoperative pain, hyperalgesia andinflammation. In fact, activated platelets have been shown to exciteperipheral nociceptors in vitro. Ringkamp, M., et. al., Activated HumanPlatelets in Plasma Excite Nociceptors in Rat Skin, In Vitro, Neurosci.Lett. 170, pp. 103-106 (1994). Similarly, histamine and bradykinin alsoare released into tissues during trauma. Kimura, E., et. al., Changes inBradykinin Level in Coronary Sinus Blood After the ExperimentalOcclusion of a Coronary Artery, Am Heart J. 85, pp. 635-647 (1973);Douglas, 1985; Dray et. al. (1993).

In addition, prostaglandins also are known to cause pain andinflammation. Cyclooxygenase inhibitors, e.g., ibuprofen, are commonlyused in non-surgical and post-operative settings to block the productionof prostaglandins, thereby reducing prostaglandin-mediated pain andinflammation. Flower, R. J., et. al., Analgesic-Antipyretics andAnti-Inflammatory Agents; Drugs Employed in the Treatment of Gout, inGoodman, L. S., et. al., ed., The Pharmacological Basis of Therapeutics,MacMillan Publishing Company, New York, pp. 674-715 (1985).Cyclooxygenase inhibitors are associated with some adverse systemic sideeffects when applied conventionally. For example, indomethacin orketorolac have well recognized gastrointestinal and renal adverse sideeffects.

As discussed, 5-HT, histamine, bradykinin and prostaglandins cause painand inflammation. The various receptors through which these agentsmediate their effects on peripheral tissues have been known and/ordebated for the past two decades. Most studies have been performed inrats or other animal models. However, there are differences inpharmacology and receptor sequences between human and animal species.There have been no studies conclusively demonstrating the importance of5-HT, bradykinin or histamine in producing postoperative pain in humans.

Furthermore, antagonists of these mediators currently are not used forpostoperative pain treatment. A class of drugs, termed 5-HT andnorepinephrine uptake antagonists, which includes amitriptyline, hasbeen used orally with moderate success for chronic pain conditions.However, the mechanisms of chronic versus acute pain states are thoughtto be considerably different. In fact, two studies in the acute painsetting using amitriptyline perioperatively have shown no pain-relievingeffect of amitriptyline. Levine, J. D., et. al., Desipramine EnhancesOpiate Postoperative Analgesia, Pain 27, pp. 45-49 (1986); Kerrick, J.M., et. al., Low-Dose Amitriptyline as an Adjunct to Opioids forPostoperative Orthopedic Pain: a Placebo-Controlled Trial Period, Pain52, pp. 325-30 (1993). In both studies the drug was given orally. Thesecond study noted that oral amitriptyline actually produced a loweroverall sense of well-being in postoperative patients, which may be dueto the drug's affinity for multiple amine receptors in the brain.

Amitriptyline, in addition to blocking the uptake of 5-HT andnorepinephrine, is a potent 5-HT receptor antagonist. Therefore, thelack of efficacy in reducing postoperative pain in thepreviously-mentioned studies would appear to conflict with the proposalof a role for endogenous 5-HT in acute pain. There are a number ofreasons for the lack of acute pain relief found with amitriptyline inthese two studies. (1) The first study (Levine et al., 1986) usedamitriptyline preoperatively for one week up until the night prior tosurgery whereas the second study (Kerrick et al., 1993) only usedamitriptyline postoperatively. Therefore, no amitriptyline was presentin the operative site tissues during the actual tissue injury phase, thetime at which 5-HT is purported to be released. (2) Amitriptyline isknown to be extensively metabolized by the liver. With oraladministration, the concentration of amitriptyline in the operative sitetissues may not have been sufficiently high for a long enough timeperiod to inhibit the activity of postoperatively released 5-HT in thesecond study. (3) Since multiple inflammatory mediators exist, andstudies have demonstrated synergism between the inflammatory mediators,blocking only one agent (5-HT) may not sufficiently inhibit theinflammatory response to tissue injury.

There have been a few studies demonstrating the ability of extremelyhigh concentrations (1%-3% solutions—i.e., 10-30 mg per milliliter) ofhistamine₁ (H₁) receptor antagonists to act as local anesthetics forsurgical procedures. This anesthetic effect is not believed to bemediated via H₁ receptors but, rather, due to a non-specific interactionwith neuronal membrane sodium channels (similar to the action oflidocaine). Given the side effects (e.g., sedation) associated withthese high “anesthetic” concentrations of histamine receptorantagonists, local administration of histamine receptor antagonistscurrently is not used in the perioperative setting.

III. SUMMARY OF THE INVENTION

The present invention provides a solution constituting a mixture ofmultiple agents in low concentrations directed at inhibiting locally themediators of pain, inflammation, spasm and restenosis in a physiologicelectrolyte carrier fluid. The invention also provides a method forperioperative delivery of the irrigation solution containing theseagents directly to a surgical site, where it works locally at thereceptor and enzyme levels to preemptively limit pain, inflammation,spasm and restenosis at the site. Due to the local perioperativedelivery method of the present invention, a desired therapeutic effectcan be achieved with lower doses of agents than are necessary whenemploying other methods of delivery (i.e., intravenous, intramuscular,subcutaneous and oral). The anti-pain/anti-inflammation agents in thesolution include agents selected from the following classes of receptorantagonists and agonists and enzyme activators and inhibitors, eachclass acting through a differing molecular mechanism of action for painand inflammation inhibition: (1) serotonin receptor antagonists; (2)serotonin receptor agonists; (3) histamine receptor antagonists; (4)bradykinin receptor antagonists; (5) kallikrein inhibitors; (6)tachykinin receptor antagonists, including neurokinin₁ and neurokinin₂receptor subtype antagonists; (7) calcitonin gene-related peptide (CGRP)receptor antagonists; (8) interleukin receptor antagonists; (9)inhibitors of enzymes active in the synthetic pathway for arachidonicacid metabolites, including (a) phospholipase inhibitors, including PLA₂isoform inhibitors and PLCγ isoform inhibitors, (b) cyclooxygenaseinhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptorantagonists including eicosanoid EP-1 and EP-4 receptor subtypeantagonists and thromboxane receptor subtype antagonists; (11)leukotriene receptor antagonists including leukotriene B₄ receptorsubtype antagonists and leukotriene D₄ receptor subtype antagonists;(12) opioid receptor agonists, including μ-opioid, δ-opioid, andκ-opioid receptor subtype agonists; (13) purinoceptor agonists andantagonists including P_(2X) receptor antagonists and P_(2Y) receptoragonists; and (14) adenosine triphosphate (ATP)-sensitive potassiumchannel openers. Each of the above agents functions either as ananti-inflammatory agent and/or as an anti-nociceptive, i.e., anti-painor analgesic, agent. The selection of agents from these classes ofcompounds is tailored for the particular application.

Several preferred embodiments of the solution of the present inventionalso include anti-spasm agents for particular applications. For example,anti-spasm agents may be included alone or in combination withanti-pain/anti-inflammation agents in solutions used for vascularprocedures to limit vasospasm, and anti-spasm agents may be included forurologic procedures to limit spasm in the urinary tract and bladderwall. For such applications, anti-spasm agents are utilized in thesolution. For example, an anti-pain/anti-inflammation agent which alsoserves as an anti-spasm agent may be included. Suitableanti-inflammatory/anti-pain agents which also act as anti-spasm agentsinclude serotonin receptor antagonists, tachykinin receptor antagonists,and ATP-sensitive potassium channel openers. Other agents which may beutilized in the solution specifically for their anti-spasm propertiesinclude calcium channel antagonists, endothelin receptor antagonists andthe nitric oxide donors (enzyme activators).

Specific preferred embodiments of the solution of the present inventionfor use in cardiovascular and general vascular procedures includeanti-restenosis agents, which most preferably are used in combinationwith anti-spasm agents. Suitable anti-restenosis agents include: (1)antiplatelet agents including: (a) thrombin inhibitors and receptorantagonists, (b) adenosine disphosphate (ADP) receptor antagonists (alsoknown as purinoceptor₁ receptor antagonists), (c) thromboxane inhibitorsand receptor antagonists and (d) platelet membrane glycoprotein receptorantagonists; (2) inhibitors of cell adhesion molecules, including (a)selectin inhibitors and (b) integrin inhibitors; (3) anti-chemotacticagents; (4) interleukin receptor antagonists (which also serve asanti-pain/anti-inflammation agents); and (5) intracellular signalinginhibitors including: (a) protein kinase C (PKC) inhibitors and proteintyrosine kinase inhibitors, (b) modulators of intracellular proteintyrosine phosphatases, (c) inhibitors of src homology₂ (SH2) domains,and (d) calcium channel antagonists. Such agents are useful inpreventing restenosis of arteries treated by angioplasty, rotationalatherectomy or other cardiovascular or general vascular therapeutic ordiagnostic procedure.

The present invention also provides a method for manufacturing amedicament compounded as a dilute irrigation solution for use incontinuously irrigating an operative site or wound during an operativeprocedure. The method entails dissolving in a physiologic electrolytecarrier fluid a plurality of anti-pain/anti-inflammatory agents, and forsome applications anti-spasm agents and/or anti-restenosis agents, eachagent included at a concentration of preferably no more than 100,000nanomolar, and more preferably no more than 10,000 nanomolar.

The method of the present invention provides for the delivery of adilute combination of multiple receptor antagonists and agonists andenzyme inhibitors and activators directly to a wound or operative site,during therapeutic or diagnostic procedures for the inhibition of pain,inflammation, spasm and restenosis. Since the active ingredients in thesolution are being locally applied directly to the operative tissues ina continuous fashion, the drugs may be used efficaciously at extremelylow doses relative to those doses required for therapeutic effect whenthe same drugs are delivered orally, intramuscularly, subcutaneously orintravenously. As used herein, the term “local” encompasses applicationoi a drug in and around a wound or other operative site, and excludesoral, subcutaneous, intravenous and intramuscular administration. Theterm “continuous” as used herein encompasses uninterrupted application,repeated application at frequent intervals (e.g., repeated intravascularboluses at frequent intervals intraprocedurally), and applications whichare uninterrupted except for brief cessations such as to permit theintroduction of other drugs or agents or procedural equipment, such thata substantially constant predetermined concentration is maintainedlocally at the wound or operative site.

The advantages of low dose applications of agents are three-fold. Themost important is the absence of systemic side effects which often limitthe usefulness of these agents. Additionally, the agents selected forparticular applications in the solutions of the present invention arehighly specific with regard to the mediators on which they work. Thisspecificity is maintained by the low dosages utilized. Finally, the costof these active agents per operative procedure is low.

The advantages of local administration of the agents via luminalirrigation or other fluid application are the following: (1) localadministration guarantees a known concentration at the target site,regardless of interpatient variability in metabolism, blood flow, etc.;(2) because of the direct mode of delivery, a therapeutic concentrationis obtained instantaneously and, thus, improved dosage control isprovided; and (3) local administration of the active agents directly toa wound or operative site also substantially reduces degradation of theagents through extracellular processes, e.g., first- and second-passmetabolism, that would otherwise occur if the agents were given orally,intravenously, subcutaneously or intramuscularly. This is particularlytrue for those active agents that are peptides, which are metabolizedrapidly. Thus, local administration permits the use of compounds oragents which otherwise could not be employed therapeutically. Forexample, some agents in the following classes are peptidic: bradykininreceptor antagonists; tachykinin receptor antagonists; opioid receptoragonists; CGRP receptor antagonists; and interleukin receptorantagonists. Local, continuous delivery to the wound or operative siteminimizes drug degradation or metabolism while also providing for thecontinuous replacement of that portion of the agent that may bedegraded, to ensure that a local therapeutic concentration, sufficientto maintain receptor occupancy, is maintained throughout the duration ofthe operative procedure.

Local administration of the solution perioperatively throughout asurgical procedure in accordance with the present invention produces apreemptive analgesic, anti-inflammatory, anti-spasmodic oranti-restenotic effect. As used herein, the term “perioperative”encompasses application intraprocedurally, pre- and intraprocedurally,intra- and postprocedurally, and pre-, intra- and postprocedurally. Tomaximize the preemptive anti-inflammatory, analgesic (for certainapplications), antispasmodic (for certain applications) andantirestenotic (for certain applications) effects, the solutions of thepresent invention are most preferably applied pre-, intra- andpostoperatively. By occupying the target receptors or inactivating oractivating targeted enzymes prior to the initiation of significantoperative trauma locally, the agents of the present solution modulatespecific pathways to preemptively inhibit the targeted pathologicprocess. If inflammatory mediators and processes are preemptivelyinhibited in accordance with the present invention before they can exerttissue damage, the benefit is more substantial than if given after thedamage has been initiated.

Inhibiting more than one inflammatory, spasm or restenosis mediator byapplication of the multiple agent solution of the present invention hasbeen shown to dramatically reduce the degree of inflammation, pain, andspasm, and theoretically should reduce restenosis. The irrigationsolutions of the present invention include combinations of drugs, eachsolution acting on multiple receptors or enzymes. The drug agents arethus simultaneously effective against a combination of pathologicprocesses, including pain and inflammation, vasospasm, smooth musclespasm and restenosis. The action of these agents is considered to besynergistic, in that the multiple receptor antagonists and inhibitoryagonists of the present invention provide a disproportionately increasedefficacy in combination relative to the efficacy of the individualagents. The synergistic action of several of the agents of the presentinvention are discussed, by way of example, below in the detaileddescriptions of those agents.

In addition to arthroscopy, the solution of the present invention mayalso be applied locally to any human body cavity or passage, operativewound, traumatic wound (e.g., burns) or in any operative/interventionalprocedure in which irrigation can be performed. These proceduresinclude, but are not limited to, urological procedures, cardiovascularand general vascular diagnostic and therapeutic procedures, endoscopicprocedures and oral, dental and periodontal procedures. As usedhereafter, the term “wound”, unless otherwise specified, is intended toinclude surgical wounds, operative/interventional sites, traumaticwounds and burns.

Used perioperatively, the solution should result in a clinicallysignificant decrease in operative site pain and inflammation relative tocurrently-used irrigation fluids, thereby decreasing the patient'spostoperative analgesic (i.e., opiate) requirement and, whereappropriate, allowing earlier patient mobilization of the operativesite. No extra effort on the part of the surgeon and operating roompersonnel is required to use the present solution relative toconventional irrigation fluids.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in greater detail, by way ofexample, with reference to the accompanying drawings in which:

FIG. 1 provides a schematic overview of a generic vascular cell showingmolecular targets and flow of signaling information leading tocontraction, secretion and/or proliferation. The integration ofextrinsic signals through receptors, ion channels and other membraneproteins are common to platelets, neutrophils, endothelial cells andsmooth muscle cells. Representative examples of molecular targets areincluded for major groups of molecules which are therapeutic targets ofdrugs included in the solutions of the present invention.

FIG. 2 provides a detailed diagram of the signaling pathwaysillustrating “crosstalk” between G-protein coupled receptor (GPCR)pathways and receptor tyrosine kinase (RTK) pathways in a vascularsmooth muscle cell. Only representative proteins in each pathway havebeen shown to simplify the flow of information. Activation of GPCRsleads to increases in intracellular calcium and increased protein kinaseC (PKC) activity and subsequent smooth muscle contraction or spasm. Inaddition, “crosstalk” to the RTK signaling pathway occurs throughactivation of PYK2 (a newly discovered protein tyrosine kinase) andPTK-X (an undefined protein tyrosine kinase), triggering proliferation.Conversely, while activation of RTKs directly initiates proliferation,“crosstalk” to the GPCR pathway occurs at the level of PKC activity andcalcium levels. LGR designates ligand-gated receptor, and MAPKdesignates mitogen-activated protein kinase. These interactions definethe basis for synergistic interactions between molecular targetsmediating spasm and restenosis. The GPCR signaling pathway also mediatessignal transduction (FIGS. 3 and 7) leading to pain transmission inother cell types (e.g., neurons).

FIG. 3 provides a diagram of the G-Protein Coupled Receptor (GPCR)pathway. Specific molecular sites of action for some drugs in apreferred arthroscopic solution of the present invention are identified.

FIG. 4 provides a diagram of the G-Protein Coupled Receptor (GPCR)pathway including the signaling proteins responsible for “crosstalk”with the Growth Factor Receptor signaling pathway. Specific molecularsites of action for some drugs in a preferred cardiovascular and generalvascular solution of the present invention are identified. (See alsoFIG. 5).

FIG. 5 provides a diagram of the Growth Factor Receptor signalingpathway including the signaling proteins responsible for “crosstalk”with the G-Protein Coupled Receptor signaling pathway. Specificmolecular sites of action for some drugs in a preferred cardiovascularand general vascular solution of the present invention are identified.(See also FIG. 4).

FIG. 6 provides a diagram of the G-Protein Coupled Receptor pathwayincluding the signaling proteins responsible for “crosstalk” with theGrowth Factor Receptor signaling pathway. Specific molecular sites ofaction for some drugs in a preferred urologic solution are identified.

FIG. 7 provides a diagram of the G-Protein Coupled Receptor pathway.Specific molecular sites of action for some drugs in a preferred generalsurgical wound solution of the present invention are identified.

FIG. 8 provides a diagram of the mechanism of action of nitric oxide(NO) donor drugs and NO causing relaxation of vascular smooth muscle.Physiologically, certain hormones and transmitters can activate a formof NO synthase in the endothelial cell through elevated intracellularcalcium resulting in increased synthesis of NO. NO donors may generateNO extracellularly or be metabolized to NO within the smooth musclecell. Extracellular NO can diffuse across the endothelial cell ordirectly enter the smooth muscle cell. The primary target of NO is thesoluble guanylate cyclase (GC), leading to activation of acGMP-dependent protein kinase (PKG) and subsequent extrusion of calciumfrom the smooth muscle cell via a membrane pump. NO also hyperpolarizesthe cell by opening potassium channels which in turn cause closure ofvoltage-sensitive calcium channels. Thus, the synergistic interactionsof calcium channel antagonists, potassium channel openers and NO donorsare evident from the above signal transduction pathway.

FIGS. 9, 10A and 10B provide charts of the percent of vasoconstrictionversus time in control arteries, in the proximal segment of subjectarteries, and in the distal segment of subject arteries, respectively,for the animal study described in EXAMPLE VII herein demonstrating theeffect on vasoconstriction of infusion with histamine and serotoninantagonists, used in the solutions of the present invention, duringballoon angioplasty.

FIGS. 11 and 12 provide charts of plasma extravasation versus dosage ofamitriptyline, used in the solutions of the present invention, deliveredintravenously and intra-articularly, respectively, to knee joints inwhich extravasation has been induced by introduction of5-hydroxytryptamine in the animal study described in EXAMPLE VIIIherein.

FIGS. 13, 14 and 15 provide charts of mean vasoconstriction (negativevalues) or vasodilation (positive values),±1 standard error of the meanfor the proximal (FIG. 13), mid (FIG. 14) and distal (FIG. 15) segmentsof arteries treated with saline (N=4) or with a solution formulated inaccordance with the present invention (N=7), at the immediate and 15minute post-rotational atherectomy time points in the animal study ofExample XIII described herein.

V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The irrigation solution of the present invention is a dilute solution ofmultiple pain/inflammation inhibitory agents, anti-spasm agents andanti-restenosis agents in a physiologic carrier. The carrier is aliquid, which as used herein is intended to encompass biocompatiblesolvents, suspensions, polymerizable and non-polymerizable gels, pastesand salves. Preferably the carrier is an aqueous solution which mayinclude physiologic electrolytes, such as normal saline or lactatedRinger's solution.

The anti-inflammation/anti-pain agents are selected from the groupconsisting of: (1) serotonin receptor antagonists; (2) serotoninreceptor agonists; (3) histamine receptor antagonists; (4) bradykininreceptor antagonists; (5) kallikrein inhibitors; (6) tachykinin receptorantagonists, including neurokinin₁ and neurokinin₂ receptor subtypeantagonists; (7) calcitonin gene-related peptide (CGRP) receptorantagonists; (8) interleukin receptor antagonists; (9) inhibitors ofenzymes active in the synthetic pathway for arachidonic acidmetabolites, including (a) phospholipase inhibitors, including PLA₂isoform inhibitors and PLCγ isoform inhibitors (b) cyclooxygenaseinhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptorantagonists including eicosanoid EP-1 and EP-4 receptor subtypeantagonists and thromboxane receptor subtype antagonists; (11)leukotriene receptor antagonists including leukotriene B₄ receptorsubtype antagonists and leukotriene D₄ receptor subtype antagonists;(12) opioid receptor agonists, including μ-opioid, δ-opioid, andκ-opioid receptor subtype agonists; (13) purinoceptor agonists andantagonists including P_(2X) receptor antagonists and P_(2Y) receptoragonists; and (14) adenosine triphosphate (ATP)-sensitive potassiumchannel openers.

Suitable anti-inflammatory/anti-pain agents which also act as anti-spasmagents include serotonin receptor antagonists, tachykinin receptorantagonists, ATP-sensitive potassium channel openers and calcium channelantagonists. Other agents which may be utilized in the solutionspecifically for their anti-spasm properties including endothelinreceptor antagonists, calcium channel antagonists and the nitric oxidedonors (enzyme activators).

Specific preferred embodiments of the solution of the present inventionfor use in cardiovascular and general vascular procedures includeanti-restenosis agents, which most preferably are used in combinationwith anti-spasm agents. Suitable anti-restenosis agents include: (1)antiplatelet agents including: (a) thrombin inhibitors and receptorantagonists, (b) adenosine disphosphate (ADP) receptor antagonists (alsoknown as purinoceptor₁ receptor antagonists), (c) thromboxane inhibitorsand receptor antagonists and (d) platelet membrane glycoprotein receptorantagonists; (2) inhibitors of cell adhesion molecules, including (a)selectin inhibitors and (b) integrin inhibitors; (3) anti-chemotacticagents; (4) interleukin receptor antagonists (which also serve asanti-pain/anti-inflammation agents); and (5) intracellular signalinginhibitors including: (a) protein kinase C (PKC) inhibitors and proteintyrosine phosphatases, (b) modulators of intracellular protein tyrosinekinase inhibitors, (c) inhibitors of src homology₂ (SH2) domains, and(d) calcium channel antagonists. Such agents are useful in preventingrestenosis of arteries treated by angioplasty, rotational atherectomy orother cardiovascular or general vascular therapeutic procedure.

In each of the surgical solutions of the present invention, the agentsare included in low concentrations and are delivered locally in lowdoses relative to concentrations and doses required with conventionalmethods of drug administration to achieve the desired therapeuticeffect. It is impossible to obtain an equivalent therapeutic effect bydelivering similarly dosed agents via other (i.e., intravenous,subcutaneous, intramuscular or oral) routes of drug administration sincedrugs given systemically are subject to first- and second-passmetabolism. The concentration of each agent is determined in part basedon its dissociation constant, K_(d). As used herein, the termdissociation constant is intended to encompass both the equilibriumdissociation constant for its respective agonist-receptor orantagonist-receptor interaction and the equilibrium inhibitory constantfor its respective activator-enzyme or inhibitor-enzyme interaction.Each agent is preferably included at a low concentration of 0.1 to10,000 times K_(d) nanomolar, except for cyclooxygenase inhibitors,which may be required at larger concentrations depending on theparticular inhibitor selected. Preferably, each agent is included at aconcentration of 1.0 to 1,000 times K_(d) nanomolar and most preferablyat approximately 100 times K_(d) nanomolar. These concentrations areadjusted as needed to account for dilution in the absence of metabolictransformation at the local delivery site. The exact agents selected foruse in the solution, and the concentration of the agents, varies inaccordance with the particular application, as described below.

A solution in accordance with the present invention can include just asingle or multiple pain/inflammation inhibitory agent(s), a single ormultiple anti-spasm agent(s), a combination of both anti-spasm andpain/inflammation inhibitory agents, or anti-restenosis agents from theenumerated classes, at low concentration. However, due to theaforementioned synergistic effect of multiple agents, and the desire tobroadly block pain and inflammation, spasm and restenosis, it ispreferred that multiple agents be utilized.

The surgical solutions constitute a novel therapeutic approach bycombining multiple pharmacologic agents acting at distinct receptor andenzyme molecular targets. To date, pharmacologic strategies have focusedon the development of highly specific drugs that are selective forindividual receptor subtypes and enzyme isoforms that mediate responsesto individual signaling neurotransmitters and hormones. As an example,endothelin peptides are some of the most potent vasoconstrictors known.Selective antagonists that are specific for subtypes of endothelin (ET)receptors are being sought by several pharmaceutical companies for usein the treatment of numerous disorders involving elevated endothelinlevels in the body. Recognizing the potential role of the receptorsubtype ET_(A) in hypertension, these drug companies specifically aretargeting the development of selective antagonists to the ET_(A)receptor subtype for the anticipated treatment of coronary vasospasm.This standard pharmacologic strategy, although well accepted, is notoptimal since many other vasoconstrictor agents (e.g., serotonin,prostaglandin, eicosanoid, etc.) simultaneously may be responsible forinitiating and maintaining a vasospastic episode (see FIGS. 2 and 4).Furthermore, despite inactivation of a single receptor subtype orenzyme, activation of other receptor subtypes or enzymes and theresultant signal transmission often can trigger a cascade effect. Thisexplains the significant difficulty in employing a singlereceptor-specific drug to block a pathophysiologic process in whichmultiple transmitters play a role. Therefore, targeting only a specificindividual receptor subtype, such as ET_(A), is likely to beineffective.

In contrast to the standard approach to pharmacologic therapy, thetherapeutic approach of the present surgical solutions is based on therationale that a combination of drugs acting simultaneously on distinctmolecular targets is required to inhibit the full spectrum of eventsthat underlie the development of a pathophysiologic state. Furthermore,instead of targeting a specific receptor subtype alone, the surgicalsolutions are composed of drugs that target common molecular mechanismsoperating in different cellular physiologic processes involved in thedevelopment of pain, inflammation, vasospasm, smooth muscle spasm andrestenosis (see FIG. 1). In this way, the cascading of additionalreceptors and enzymes in the nociceptive, inflammatory, spasmodic andrestenotic pathways is minimized by the surgical solutions. In thesepathophysiologic pathways, the surgical solutions inhibit the cascadeeffect both “upstream” and “downstream”.

An example of “upstream” inhibition is the cyclooxygenase antagonists inthe setting of pain and inflammation. The cyclooxygenase enzymes (COX₁and COX₂) catalyze the conversion of arachidonic acid to prostaglandin Hwhich is an intermediate in the biosynthesis of inflammatory andnociceptive mediators including prostaglandins, leukotrienes, andthromboxanes. The cyclooxygenase inhibitors block “upstream” theformation of these inflammatory and nociceptive mediators. This strategyprecludes the need to block the interactions of the seven describedsubtypes of prostanoid receptors with their natural ligands. A similar“upstream” inhibitor included in the surgical solutions is aprotinin, akallikrein inhibitor. The enzyme kallikrein, a serine protease, cleavesthe high molecular weight kininogens in plasma to produce bradykinins,important mediators of pain and inflammation. By inhibition ofkallikrein, aprotinin effectively inhibits the synthesis of bradykinins,thereby providing an effective “upstream” inhibition of theseinflammatory mediators.

The surgical solutions also make use of “downstream” inhibitors tocontrol the pathophysiologic pathways. In vascular smooth musclepreparations that have been precontracted with a variety ofneurotransmitters (e.g., serotonin, histamine, endothelin, andthromboxane) implicated in coronary vasospasm, ATP-sensitive potassiumchannel openers (KCOs) produce smooth muscle relaxation which isconcentration dependent (Quast et al., 1994; Kashiwabara et al., 1994).The KCOs, therefore, provide a significant advantage to the surgicalsolutions in the settings of vasospasm and smooth muscle spasm byproviding “downstream” antispasmodic effects that are independent of thephysiologic combination of agonists initiating the spasmodic event (seeFIGS. 2 and 4). Similarly, NO donors and voltage-gated calcium channelantagonists can limit vasospasm and smooth muscle spasm initiated bymultiple mediators known to act earlier in the spasmodic pathway.

The following is a description of suitable drugs falling in theaforementioned classes of anti-inflammation/anti-pain agents, as well assuitable concentrations for use in solutions, of the present invention.While not wishing to be limited by theory, the justification behind theselection of the various classes of agents which is believed to renderthe agents operative is also set forth.

A. Serotonin Receptor Antagonists

Serotonin (5-HT) is thought to produce pain by stimulating serotonin₂(5-HT₂) and/or serotonin₃ (5-HT₃) receptors on nociceptive neurons inthe periphery. Most researchers agree that 5-HT₃ receptors on peripheralnociceptors mediate the immediate pain sensation produced by 5-HT(Richardson et al., 1985). In addition to inhibiting 5-HT-induced pain,5-HT₃ receptor antagonists, by inhibiting nociceptor activation, alsomay inhibit neurogenic inflammation. Barnes P. J., et. al., Modulationof Neurogenic Inflammation: Novel Approaches to Inflammatory Disease,Trends in Pharmacological Sciences 11, pp. 185-189 (1990). A study inrat ankle joints, however, claims the 5-HT₂ receptor is responsible fornociceptor activation by 5-HT. Grubb, B. D., et. al., A Study of5-HT-Receptors Associated with Afferent Nerves Located in Normal andInflamed Rat Ankle Joints, Agents Actions 25, pp.216-18(1988).Therefore, activation of 5-HT₂ receptors also may play a role inperipheral pain and neurogenic inflammation.

One goal of the solution of the present invention is to block pain and amultitude of inflammatory processes. Thus, 5-HT₂ and 5-HT₃ receptorantagonists are both suitably used, either individually or together, inthe solution of the present invention, as shall be describedsubsequently. Amitriptyline (Elavil™) is a suitable 5-HT₂ receptorantagonist for use in the present invention. Amitriptyline has been usedclinically for numerous years as an anti-depressant, and is found tohave beneficial effects in certain chronic pain patients. Metoclopramide(Reglan™) is used clinically as an anti-emetic drug, but displaysmoderate affinity for the 5-HT₃ receptor and can inhibit the actions of5-HT at this receptor, possibly inhibiting the pain due to 5-HT releasefrom platelets. Thus, it also is suitable for use in the presentinvention.

Other suitable 5-HT₂ receptor antagonists include imipramine, trazodone,desipramine and ketanserin. Ketanserin has been used clinically for itsanti-hypertensive effects. Hedner, T., et. al., Effects of a NewSerotonin Antagonist, Ketanserin, in Experimental and ClinicalHypertension, Am J of Hypertension, pp.317s-23s (July 1988). Othersuitable 5-HT₃ receptor antagonists include cisapride and ondansetron.The cardiovascular and general vascular solution also may contain aserotonin_(1B) (also known as serotonin_(1Dβ)) antagonist becauseserotonin has been shown to produce significant vascular spasm viaactivation of the serotonin_(1B) receptors in humans. Kaumann, A. J., etal., Variable Participation of 5-HT1-Like Receptors and 5-HT2 Receptorsin Serotonin-Induced Contraction of Human Isolated Coronary Arteries,Circulation 90, pp. 1141-53 (1994). Suitable serotonin_(1B) receptorantagonists include yohimbine,N-[-methoxy-3-(4-methyl-1-piperanzinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2, 4-oxadiazol-3-yl)[1, 1-biphenyl]-4-carboxamide (“GR127935”) andmethiothepin. Therapeutic and preferred concentrations for use of thesedrugs in the solution of the present invention are set forth in Table 1.

TABLE 1 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Serotonin₂ Receptor Antagonists:amitriptyline 0.1-1,000 50-500 imipramine 0.1-1,000 50-500 trazodone0.1-2,000 50-500 desipramine 0.1-1,000 50-500 ketanserin 0.1-1,00050-500 Serotonin₃ Receptor Antagonists: tropisetron 0.01-100   0.05-50  metoclopramide  10-10,000  200-2,000 cisapride 0.1-1,000 20-200ondansetron 0.1-1,000 20-200 Serotonin_(1B) (Human 1D_(β)) Antagonists:yohimbine 0.1-1,000 50-500 GR127935 0.1-1,000 10-500 methiothepin0.1-500    1-100

B. Serotonin Receptor Agonists

5-HT_(1A), 5-HT_(1B) and 5-HT_(1D) receptors are known to inhibitadenylate cyclase activity. Thus including a low dose of theseserotonin_(1A), serotonin_(1B) and serotonin_(1D) receptor agonists inthe solution should inhibit neurons mediating pain and inflammation. Thesame action is expected from serotonin_(1E) and serotonin_(1F) receptoragonists because these receptors also inhibit adenylate cyclase.

Buspirone is a suitable 1A receptor agonist for use in the presentinvention. Sumatriptan is a suitable 1A, 1B, 1D and 1F receptor agonist.A suitable 1B and 1D receptor agonist is dihydroergotamine. A suitable1E receptor agonist is ergonovine. Therapeutic and preferredconcentrations for these receptor agonists are provided in Table 2.

TABLE 2 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Serotonin_(1A) Agonists:buspirone 1-1,000 10-200 sumatriptan 1-1,000 10-200 Serotonin_(1B)Agonists: dihydroergotamine 0.1-1,000   10-100 sumatriptan 1-1,00010-200 Serotonin_(1D) Agonists: dihydroergotamine 0.1-1,000   10-100sumatriptan 1-1,000 10-200 Serotonin_(1E) Agonists: ergonovine 10-2,000  100-1,000 Serotonin_(1F) Agonists: sumatriptan 1-1,000 10-200

C. Histamine Receptor Antagonists

Histamine receptors generally are divided into histamine₁ (H₁) andhistamine₂ (H₂) subtypes. The classic inflammatory response to theperipheral administration of histamine is mediated via the H₁ receptor.Douglas, 1985. Therefore, the solution of the present inventionpreferably includes a histamine H₁ receptor antagonist. Promethazine(Phenergan™) is a commonly used anti-emetic drug which potently blocksH₁ receptors, and is suitable for use in the present invention.Interestingly, this drug also has been shown to possess local anestheticeffects but the concentrations necessary for this effect are severalorders higher than that necessary to block H₁ receptors, thus, theeffects are believed to occur by different mechanisms. The histaminereceptor antagonist concentration in the solution is sufficient toinhibit H₁ receptors involved in nociceptor activation, but not toachieve a “local anesthetic” effect, thereby eliminating the concernregarding systemic side effects.

Histamine receptors also are known to mediate vasomotor tone in thecoronary arteries. In vitro studies in the human heart have demonstratedthat the histamine₁ receptor subtype mediates contraction of coronarysmooth muscle. Ginsburg, R., et al., Histamine Provocation of ClinicalCoronary Artery Spasm: Implications Concerning Pathogenesis of VariantAngina Pectoris, American Heart J., Vol. 102, pp. 819-822, (1980). Somestudies suggest that histamine-induced hypercontractility in the humancoronary system is most pronounced in the proximal arteries in thesetting of atherosclerosis and the associated denudation of the arterialendothelium. Keitoku, M. et al., Different Histamine Actions in Proximaland Distal Human Coronary Arteries in Vitro, Cardiovascular Research 24,pp. 614-622, (1990). Therefore, histamine receptor antagonists may beincluded in the cardiovascular irrigation solution.

Other suitable H₁ receptor antagonists include terfenadine,diphenhydramine, amitriptyline, mepyramine and tripolidine. Becauseamitriptyline is also effective as a serotonin₂ receptor antagonist, ithas a dual function as used in the present invention. Suitabletherapeutic and preferred concentrations for each of these H₁ receptorantagonists are set forth in Table 3.

TABLE 3 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Histamine₁ Receptor Antagonists:promethazine 0.1-1,000 50-200 diphenhydramine 0.1-1,000 50-200amitriptyline 0.1-1,000 50-500 terfenadine 0.1-1,000 50-500 mepyramine(pynlainine) 0.1-1,000  5-200 tripolidine 0.01-100   5-20

D. Bradykinin Receptor Antagonists

Bradykinin receptors generally are divided into bradykinin₁ (B₁) andbradykinin₂ (B₂) subtypes. Studies have shown that acute peripheral painand inflammation produced by bradykinin are mediated by the B2 subtypewhereas bradykinin-induced pain in the setting of chronic inflammationis mediated via the B₁ subtype. Perkins, M. N., et. al., AntinociceptiveActivity of the Bradykinin B1 and B2 Receptor Antagonists, des-Arg⁹,[Leu⁸]-BK and HOE 140, in Two Models of Persistent Hyperalgesia in theRat, Pain 53, pp. 191-97 (1993); Dray, A., et. al., Bradykinin andInflammatory Pain, Trends Neurosci 16, pp. 99-104 (1993), each of whichreferences is hereby expressly incorporated by reference.

At present, bradykinin receptor antagonists are not used clinically.These drugs are peptides (small proteins), and thus they cannot be takenorally, because they would be digested. Antagonists to B₂ receptorsblock bradykinin-induced acute pain and inflammation. Dray et. al.,1993. B₁ receptor antagonists inhibit pain in chronic inflammatoryconditions. Perkins et al., 1993; Dray et. al., 1993. Therefore,depending on the application, the solution of the present inventionpreferably includes either or both bradykinin B₁ and B₂ receptorantagonists. For example, arthroscopy is performed for both acute andchronic conditions, and thus an irrigation solution for arthroscopycould include both B₁ and B₂ receptor antagonists.

Suitable bradykinin receptor antagonists for use in the presentinvention include the following bradykinin₁ receptor antagonists: the[des-Arg¹⁰] derivative of D-Arg-(Hyp³-Thi⁵-D-Tic⁷-Oic⁸)-BK (“the[des-Arg¹⁰] derivative of HOE 140”, available from HoechstPharmaceuticals); and [Leu⁸] des-Arg⁹-BK. Suitable bradykinin₂ receptorantagonists include: [D-Phe⁷]-BK; D-Arg-(Hyp³-Thi^(5,8)-D-Phe⁷)-BK (“NPC349”); D-Arg-(Hyp³—D-Phe⁷)-BK (“NPC 567”); andD-Arg-(Hyp³-Thi⁵-D-Tic⁷-Oic⁸)-BK (“HOE 140”). These compounds are morefully described in the previously incorporated Perkins et. al. 1993 andDray et. al. 1993 references. Suitable therapeutic and preferredconcentrations are provided in Table 4.

TABLE 4 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Bradykinin₁ Receptor Antagonists:[Leu⁸] des-Arg⁹-BK 1-1,000 50-500 [des-Arg¹⁰] derivative of HOE 1401-1,000 50-500 [leu⁹ ] [des-Arg¹⁰] kalliden 0.1-500   10-200 Bradykinin₂Receptor Antagonists: [D-Phe⁷]-BK 100-10,000   200-5,000 NPC 349 1-1,00050-500 NPC 567 1-1,000 50-500 HOE 140 1-1,000 50-500

E. Kallikrein Inhibitors

The peptide bradykinin is an important mediator of pain andinflammation, as noted previously. Bradykinin is produced as a cleavageproduct by the action of kallikrein on high molecular weight kininogensin plasma. Therefore kallikrein inhibitors are believed to betherapeutic in inhibiting bradykinin production and resultant pain andinflammation. A suitable kallikrein inhibitor for use in the presentinvention is aprotinin. Suitable concentrations for use in the solutionsof the present invention are set forth below in Table 5.

TABLE 5 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Kallikrein Inhibitor: 0.1-1,00050-500 Aprotinin

F. Tachykinin Receptor Antagonists

Tachykinins (TKs) are a family of structurally related peptides thatinclude substance P, neurokinin A (NKA) and neurokinin B (NKB). Neuronsare the major source of TKs in the periphery. An important generaleffect of TKs is neuronal stimulation, but other effects includeendothelium-dependent vasodilation, plasma protein extravasation, mastcell recruitment and degranulation and stimulation of inflammatorycells. Maggi, C. A., Gen. Pharmacol., Vol. 22, pp. 1-24 (1991). Due tothe above combination of physiological actions mediated by activation ofTK receptors, targeting of TK receptors is a reasonable approach for thepromotion of analgesia and the treatment of neurogenic inflammation.

1. Neurokinin₁ Receptor Subtype Antagonists

Substance P activates the neurokinin receptor subtype referred to asNK₁. Substance P is an undecapeptide that is present in sensory nerveterminals. Substance P is known to have multiple actions which produceinflammation and pain in the periphery after C-fiber activation,including vasodilation, plasma extravasation and degranulation of mastcells. Levine, J. D., et. al., Peptides and the Primary AfferentNociceptor, J. Neurosci. 13, p. 2273 (1993). A suitable Substance Pantagonist is([D-Pro⁹[spiro-gamrna-lactam]Leu¹⁰,Trp¹¹]physalaemin-(1-11)) (“GR82334”). Other suitable antagonists for use in the present inventionwhich act on the NK₁ receptor are:1-imino-2-(2-methoxy-phenyl)-ethyl)-7,7-diphenyl-4-perhydroisoindolone(3aR,7aR)(“RP 67580”); and2S,3S-cis-3-(2-methoxybenzylamino)-2-benzhydrylquinuclidine (“CP96,345”). Suitable concentrations for these agents are set forth inTable 6.

TABLE 6 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Neurokinin₁ Receptor SubtypeAntagonists GR 82334 1-1,000 10-500  CP 96,345  1-10,000 100-1,000 RP67580 0.1-1,000   100-1,000

2. Neurokinin₂ Receptor Subtype Antagonists

Neurokinin A is a peptide which is colocalized in sensory neurons withsubstance P and which also promotes inflammation and pain. Neurokinin Aactivates the specific neurokinin receptor referred to as NK₂.Edmonds-Alt, S., et. al., A Potent and Selective Non-Peptide Antagonistof the Neurokinin A (NK₂) Receptor, Life Sci. 50:PL101 (1992). In theurinary tract, TKs are powerful spasmogens acting through only the NK₂receptor in the human bladder, as well as the human urethra and ureter.Maggi, C. A., Gen. Pharmacol., Vol. 22, pp. 1-24 (1991). Thus, thedesired drugs for inclusion in a surgical solution for use in urologicalprocedures would contain an antagonist to the NK₂ receptor to reducespasm. Examples of suitable NK₂ antagonists include:((S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide(“(±)-SR 48968”); Met-Asp-Trp-Phe-Dap-Leu (“MEN 10,627”); andcyc(Gln-Trp-Phe-Gly-Leu-Met) (“L 659,877”). Suitable concentrations ofthese agents are provided in Table 7.

TABLE 7 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Neurokinin₂ Receptor SubtypeAntagonists: MEN 10,627 1-1,000 10-1,000 L 659,877 10-10,000 100-10,000(±)-SR 48968 10-10,000 100-10,000

G. CGRP Receptor Antagonists

Calcitonin gene-related peptide (CGRP) is a peptide which is alsocolocalized in sensory neurons with substance P, and which acts as avasodilator and potentiates the actions of substance P. Brain, S. D.,et. al., Inflammatory Oedema Induced by Synergism Between CalcitoninGene-Related Peptide (CGRP) and Mediators of Increased VascularPermeability, Br. J. Pharmacol. 99, p. 202 (1985). An example of asuitable CGRP receptor antagonist is α-CGRP-(8-37), a truncated versionof CGRP. This polypeptide inhibits the activation of CGRP receptors.Suitable concentrations for this agent are provided in Table 8.

TABLE 8 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) CGRP Receptor Antagonist: 1-1,00010-500 α-CGRP-(8-37)

H. Interleukin Receptor Antagonist

Interleukins are a family of peptides, classified as cytokines, producedby leukocytes and other cells in response to inflammatory mediators.Interleukins (IL) may be potent hyperalgesic agents peripherally.Ferriera, S. H., et. al., Interleukin-1βas a Potent Hyperalgesic AgentAntagonized by a Tripeptide Analogue, Nature 334, p. 698 (1988). Anexample of a suitable IL-1β receptor antagonist is Lys-D-Pro-Thr, whichis a truncated version of IL-1β. This tripeptide inhibits the activationof IL-1β receptors. Suitable concentrations for this agent are providedin Table 9.

TABLE 9 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Interleukin Receptor Antagonist:1-1,000 10-500 Lys-D-Pro-Thr

I. Inhibitors of Enzymes Active in the Synthetic Pathway for ArachidonicAcid Metabolites

1. Phospholipase Inhibitors

The production of arachidonic acid by phospholipase A₂ (PLA₂) results ina cascade of reactions that produces numerous mediators of inflammation,know as eicosanoids. There are a number of stages throughout thispathway that can be inhibited, thereby decreasing the production ofthese inflammatory mediators. Examples of inhibition at these variousstages are given below.

Inhibition of the enzyme PLA₂ isoform inhibits the release ofarachidonic acid from cell membranes, and therefore inhibits theproduction of prostaglandins and leukotrienes resulting in decreasedinflammation and pain. Glaser, K. B., Regulation of Phospholipase A2Enzymes: Selective Inhibitors and Their Pharmacological Potential, Adv.Pharmacol. 32, p. 31 (1995). An example of a suitable PLA₂ isoforminhibitor is manoalide. Suitable concentrations for this agent areincluded in Table 10. Inhibition of the phospholipase C_(γ) (PLC_(γ))isoform also will result in decreased production of prostanoids andleukotrienes, and, therefore, will result in decreased pain andinflammation. An example of a PLC_(γ) isoform inhibitor is1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

TABLE 10 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) PLA₂ Isoform Inhibitor: manoalide100-100,000 500-10,000

2. Cyclooxygenase Inhibitors

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used asanti-inflammatory, anti-pyretic, anti-thrombotic and analgesic agents.Lewis, R. A., Prostaglandins and Leukotrienes, In: Textbook ofRheumatology, 3d ed. (Kelley W. N., et. al., eds.), p. 258 (1989). Themolecular targets for these drugs are type I and type II cyclooxygenases(COX-1 and COX-2). These enzymes are also known as Prostaglandin HSynthase (PGHS)-1 (constitutive) and -2 (inducible), and catalyze theconversion of arachidonic acid to Prostaglandin H which is anintermediate in the biosynthesis of prostaglandins and thromboxanes. TheCOX-2 enzyme has been identified in endothelial cells, macrophages, andfibroblasts. This enzyme is induced by IL-1 and endotoxin, and itsexpression is upregulated at sites of inflammation. Constitutiveactivity of COX-1 and induced activity of COX-2 both lead to synthesisof prostaglandins which contribute to pain and inflammation.

NSAIDs currently on the market (diclofenac, naproxen, indomethacin,ibuprofen, etc.) are generally nonselective inhibitors of both isoformsof COX, but may show greater selectively for COX-1 over COX-2, althoughthis ratio varies for the different compounds. Use of COX-1 and 2inhibitors to block formation of prostaglandins represents a bettertherapeutic strategy than attempting to block interactions of thenatural ligands with the seven described subtypes of prostanoidreceptors. Reported antagonists of the eicosanoid receptors (EP-1, EP-2,EP-3) are quite rare and only specific, high affinity antagonists of thethromboxane A2 receptor have been reported. Wallace, J. and Cirino, G.Trends in Pharm. Sci., Vol. 15 pp. 405-406 (1994).

The oral, intravenous or intramuscular use of cyclooxygenase inhibitorsis contraindicated in patients with ulcer disease, gastritis or renalimpairment. In the United States, the only available injectable form ofthis class of drugs is ketorolac (Toradol™), available from SyntexPharmaceuticals, which is conventionally used intramuscularly orintravenously in postoperative patients but, again, is contraindicatedfor the above-mentioned categories of patients. The use of ketorolac, orany other cyclooxygenase inhibitor(s), in the solution in substantiallylower dosages than currently used perioperatively may allow the use ofthis drug in otherwise contraindicated patients. The addition of acyclooxygenase inhibitor to the solutions of the present invention addsa distinct mechanism for inhibiting the production of pain andinflammation during arthroscopy or other therapeutic or diagnosticprocedure.

Preferred cyclooxygenase inhibitors for use in the present invention areketerolac and indomethacin. Of these two agents, indomethacin is lesspreferred because of the relatively high dosages required. Therapeuticand preferred concentrations for use in the solution are provided inTable 11.

TABLE 11 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Cyclooxygenase Inhibitors:ketorolac  100-10,000  500-5,000 indomethacin 1,000-500,00010,000-200,000

3. Lipooxygenase Inhibitors

Inhibition of the enzyme lipooxygenase inhibits the production ofleukotrienes, such as leukotriene B₄, which is known to be an importantmediator of inflammation and pain. Lewis, R. A., Prostaglandins andLeukotrienes, In: Textbook of Rheumatology, 3d ed. (Kelley W. N., et.al., eds.), p. 258 (1989). An example of a 5-lipooxygenase antagonist is2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (“AA861”), suitable concentrations for which are listed in Table 12.

TABLE 12 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Lipooxygenase Inhibitor:100-10,000 500-5,000 AA 861

J. Prostanoid Receptor Antagonists

Specific prostanoids produced as metabolites of arachidonic acid mediatetheir inflammatory effects through activation of prostanoid receptors.Examples of classes of specific prostanoid antagonists are theeicosanoid EP-1 and EP-4 receptor subtype antagonists and thethromboxane receptor subtype antagonists. A suitable prostaglandin E₂receptor antagonist is8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-arboxylic acid,2-acetylhydrazide (“SC 19220”). A suitable thromboxane receptor subtypeantagonist is [15-[1 α, 2β(5Z), 3β, 4α]-7-[3-[2-(phenylamino)-carbonyl]hydrazino] methyl]-7-oxobicyclo-[2,2,1]-hept-2-yl]-5-heptanoic acid (“SQ29548”). Suitable concentrations for these agents are set forth in Table13.

TABLE 13 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Eicosanoid EP-1 Antagonist:100-10,000 500-5,000 SC 19220

K. Leukotriene Receptor Antagonists

The leukotrienes (LTB₄, LTC₄, and LTD₄) are products of the5-lipooxygenase pathway of arachidonic acid metabolism that aregenerated enzymatically and have important biological properties.Leukotrienes are implicated in a number of pathological conditionsincluding inflammation. Specific antagonists are currently being soughtby many pharmaceutical companies for potential therapeutic interventionin these pathologies. Halushka, P. V., et al., Annu. Rev. Pharmacol.Toxicol. 29: 213-239 (1989); Ford-Hutchinson, A. Crit. Rev. Immunol. 10:1-12 (1990). The LTB₄ receptor is found in certain immune cellsincluding eosinophils and neutrophils. LTB₄ binding to these receptorsresults in chemotaxis and lysosomal enzyme release thereby contributingto the process of inflammation. The signal transduction processassociated with activation of the LTB₄ receptor involvesG-protein-mediated stimulation of phosphotidylinositol (PI) metabolismand elevation of intracellular calcium (see FIG. 2).

An example of a suitable leukotriene B₄ receptor antagonist is SC(+)-(S)-7-(3-(2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)-carbonyl]phenoxy(propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoicacid (“SC 53228”). Concentrations for this agent that are suitable forthe practice of the present invention are provided in Table 14. Othersuitable leukotriene B₄ receptor antagonists include[3-[-2(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino-3-oxopropyl)thio] methyl]thiopropanoic acid (“MK0571”) and the drugs LY 66,071 and ICI 20,3219. MK 0571 also acts as aLTD₄ receptor subtype antagonist.

TABLE 14 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Leukotriene B₄ Antagonist:100-10,000 500-5,000 SC 53228

L. Opioid Receptor Agonists

Activation of opioid receptors results in anti-nociceptive effects and,therefore, agonists to these receptors are desirable. Opioid receptorsinclude the μ, δ- and κ-opioid receptor subtypes. The μ-receptors arelocated on sensory neuron terminals in the periphery and activation ofthese receptors inhibits sensory neuron activity. Basbaum, A. I., et.al., Opiate analgesia: How Central is a Peripheral Target?, N. Engl. J.Med., 325:1168 (1991). δ- and κ-receptors are located on sympatheticefferent terminals and inhibit the release of prostaglandins, therebyinhibiting pain and inflammation. Taiwo, Y. O., et. al., Kappa- andDelta-Opioids Block Sympathetically Dependent Hyperalgesia, J.Neurosci., Vol. 11, page 928 (1991). The opioid receptor subtypes aremembers of the G-protein coupled receptor superfamily. Therefore, allopioid receptor agonists interact and initiate signaling through theircognate G-protein coupled receptor (see FIGS. 3 and 7). Examples ofsuitable μ-opioid receptor agonists are fentanyl andTry-D-Ala-Gly-[N-MePhe]-NH(CH₂)-OH (“DAMGO”). An example of a suitableδ-opioid receptor agonist is [D-Pen²,D-Pen⁵]enkephalin (“DPDPE”). Anexample of a suitable κ-opioid receptor agonist is(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidnyl)cyclohexyl]-benzeneacetamide (“U50,488”). Suitable concentrations for each of these agentsare set forth in Table 15.

TABLE 15 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) μ-Opioid Agonist: DAMGO 0.1-1000.5-20  sufentanyl 0.01-50   1-20 fentanyl 0.1-500  10-200 PL 0170.05-50  0.25-10   δ-Opioid Agonist: DPDPE 0.1-500  1.0-100 κ-OpioidAgonist: U50,488 0.1-500  1.0-100

M. Purinoceptor Antagonists and Agonists

Extracellular ATP acts as a signaling molecule through interactions withP₂ purinoceptors. One major class of purinoceptors are the P_(2x)purinoceptors which are ligand-gated ion channels possessing intrinsicion channels permeable to Na⁺, K⁺, and Ca²⁺. P_(2x) receptors describedin sensory neurons are important for primary afferent neurotransmissionand nociception. ATP is known to depolarize sensory neurons and plays arole in nociceptor activation since ATP released from damaged cellsstimulates P_(2X) receptors leading to depolarization of nociceptivenerve-fiber terminals. The P2X₃ receptor has a highly restricteddistribution (Chen, C. C., et. al., Nature, Vol. 377, pp. 428-431(1995)) since it is selectively expressed in sensory C-fiber nerves thatrun into the spinal cord and many of these C-fibers are known to carrythe receptors for painful stimuli. Thus, the highly restrictedlocalization of expression for the P2X₃ receptor subunits make thesesubtypes excellent targets for analgesic action (see FIGS. 3 and 7).

Suitable antagonists of P_(2X)/ATP purinoceptors for use in the presentinvention include, by way of example, suramin andpyridoxylphosphate-6-azophenyl-2,4-disulfonic acid (“PPADS”). Suitableconcentrations for these agents are provided in Table 16.

Agonists of the P_(2Y) receptor, a G-protein coupled receptor, are knownto effect smooth muscle relaxation through elevation of inositoltriphosphate (IP₃) levels with a subsequent increase in intracellularcalcium. An example of a P_(2Y) receptor agonist is 2-me-S-ATP.

TABLE 16 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Purinoceptor Antagonists: suramin100-100,000 10,000-100,000 PPADS 100-100,000 10,000-100,000

N. Adenosine Triphosphate (ATP)-Sensitive Potassium Channel Openers

ATP-sensitive potassium channels have been discovered in numeroustissues, including vascular and non-vascular smooth muscle and brain,and binding studies using radiolabeled ligands have confirmed theirexistence. Opening of these channels causes potassium (K⁺) efflux andhyperpolarizes the cell membrane (see FIG. 2). This hyperpolarizationinduces a reduction in intracellular free calcium through inhibition ofvoltage-dependent calcium (Ca²⁺) channels and receptor operated Ca²⁺channels. These combined actions drive the cell (e.g., smooth musclecell) into a relaxed state or one which is more resistant to activationand, in the case of vascular smooth muscle, results in vasorelaxation.K⁺ channel openers (KCOs) have been characterized as having potentantihypertensive activity in vivo and vasorelaxant activity in vitro(see FIG. 4). K⁺ channel openers (KCOs) also have been shown to preventstimulus coupled secretion and are considered to act on rejunctionalneuronal receptors and thus will inhibit effects due to nervestimulation and release of inflammatory mediators. Quast, U., et. al.,Cellular Pharmacology of Potassium Channel Openers in Vascular SmoothMuscle, Cardiovasc. Res., Vol. 28, pp. 805-810 (1994).

Synergistic interactions between endothelin (ET_(A)) antagonists andopeners of ATP-sensitive potassium channels (KCOs) are expected inachieving vasorelaxation or smooth muscle relaxation. A rationale fordual use is based upon the fact that these drugs have differentmolecular mechanisms of action in promoting relaxation of smooth muscleand prevention of vasospasm. An initial intracellular calcium elevationin smooth muscle cells induced by the ET_(A) receptor subsequentlytriggers activation of voltage-dependent channels and the entry ofextracellular calcium which is required for contraction. Antagonists ofthe ET_(A) receptor will specifically block this receptor mediatedeffect but not block increases in calcium triggered by activation ofother G-protein coupled receptors on the muscle cell.

Potassium-channel opener drugs, such as pinacidil, will open thesechannels causing K⁺ efflux and hyperpolarization of the cell membrane.This hyperpolarization will act to reduce contraction mediated by otherreceptors by the following mechanisms: (1) it will induce a reduction inintracellular free calcium through inhibition of voltage-dependent Ca²⁺channels by reducing the probability of opening L-type or T-type calciumchannels, (2) it will restrain agonist induced (receptor operatedchannels) Ca²⁺ release from intracellular sources through inhibition ofinositol triphosphate (IP₃) formation, and (3) it will lower theefficiency of calcium as an activator of contractile proteins.Consequently, combined actions of these two classes of drugs will clampthe target cells into a relaxed state or one which is more resistant toactivation.

Suitable ATP-sensitive K⁺ channel openers for the practice of thepresent invention include: (−)pinacidil; cromakalim; nicorandil;minoxidil; N-cyano-N′-[1,1-dimethyl-[2,2,3,3-³H]propyl]-N″-(3-pyridinyl)guanidine (“P 1075”); andN-cyano-N′-(2-nitroxyethyl)-3-pyridinecarboximidamidemonomethansulphonate (“KRN 2391”).

Concentrations for these agents are set forth in Table 17.

TABLE 17 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) ATP-Sensitive K^(±) ChannelOpener: cromakalim 10-10,000 100-10,000 nicorandil 10-10,000 100-10,000minoxidil 10-10,000 100-10,000 P 1075 0.1-1,000   10-1,000 KRN 2391 1-10,000 100-1,000  (−)pinacidil  1-10,000 100-1,000 

I. Anti-Spasm Agents

1. Multifunction Agents

Several of the anti-pain/anti-inflammatory agents described above alsoserve to inhibit vasoconstriction or smooth muscle spasm. As such, theseagents also perform the function of anti-spasm agents, and thus arebeneficially used in vascular and urologic applications.Anti-inflammatory/anti-pain agents that also serve as anti-spasm agentsinclude: serotonin receptor antagonists, particularly, serotonin₂antagonists; tachykinin receptor antagonists and ATP-sensitive potassiumchannel openers.

2. Nitric Oxide Donors

Nitric oxide donors may be included in the solutions of the presentinvention particularly for their anti-spasm activity. Nitric oxide (NO)plays a critical role as a molecular mediator of many physiologicalprocesses, including vasodilation and regulation of normal vasculartone. Within endothelial cells, an enzyme known as NO synthase (NOS)catalyzes the conversion of L-arginine to NO which acts as a diffusiblesecond messenger and mediates responses in adjacent smooth muscle cells(see FIG. 8). NO is continuously formed and released by the vascularendothelium under basal conditions which inhibits contractions andcontrols basal coronary tone and is produced in the endothelium inresponse to various agonists (such as acetylcholine) and otherendothelium dependent vasodilators. Thus, regulation of NO synthaseactivity and the resultant levels of NO are key molecular targetscontrolling vascular tone (see FIG. 8). Muramatsu, K., et. al., Coron.Artery Dis., Vol. 5, pp. 815-820 (1994).

Synergistic interactions between NO donors and openers of ATP-sensitivepotassium channels (KCOS) are expected to achieve vasorelaxation orsmooth muscle relaxation. A rationale for dual use is based upon thefact that these drugs have different molecular mechanisms of action inpromoting relaxation of smooth muscle and prevention of vasospasm. Thereis evidence from cultured coronary arterial smooth muscle cells that thevasoconstrictors: vasopressin, angotensin II and endothelin, all inhibitK_(ATP) currents through inhibition of protein kinase A. In addition, ithas been reported that K_(ATP) current in bladder smooth muscle isinhibited by muscarinic agonists. The actions of NO in mediating smoothmuscle relaxation occur via independent molecular pathways (describedabove) involving protein kinase G (see FIG. 8). This suggests that thecombination of the two classes of agents will be more efficacious inrelaxing smooth muscle than employing a single class of agent alone.

Suitable nitric oxide donors for the practice of the present inventioninclude nitroglycerin, sodium nitroprusside, the drug FK 409, FR 144420,3-morpholinosydnonimine , or linsidomine chlorohydrate, (“SIN-1”); andS-nitroso-N-acetylpenicillamine (“SNAP”). Concentrations for theseagents are set forth in Table 18.

TABLE 18 Therapeutic and Preferred Concentrations of Spasm InhibitoryAgents Therapeutic Preferred Concentrations Concentrations Class ofAgent (Nanomolar) (Nanomolar) Nitric Oxide Donors: Nitroglycerin10-10,000 100-1,000 sodium nitroprusside 10-10,000 100-1,000 SIN-110-10,000 100-1,000 SNAP 10-10,000 100-1,000 FK 409 (NOR-3) 1-1,00010-500  FR 144420 (NOR4) 10-10,000 100-5,000

3. Endothelin Receptor Antagonists

Endothelin is a 21 amino acid peptide that is one of the most potentvasoconstrictors known. Three different human endothelin peptides,designated ET-1, ET-2 and ET-3 have been described which mediate theirphysiological effects through at least two receptor subtypes referred toas ET_(A) and ET_(B) receptors. The heart and vascular smooth musclecontain predominantly ET_(A) receptors and this subtype is responsiblefor contraction in these tissues. Furthermore, ET_(A) receptors haveoften been found to mediate contractile responses in isolated smoothmuscle preparations. Antagonists of ET_(A) receptors have been found tobe potent antagonists of human coronary artery contractions. Thus,antagonists to the ET_(A) receptor should be therapeutically beneficialin the perioperative inhibition of coronary vasospasm and mayadditionally be useful in inhibition of smooth muscle contraction inurological applications. Miller, R. C., et. al., Trends in Pharmacol.Sci., Vol. 14, pp. 54-60 (1993).

Suitable endothelin receptor antagonists include:cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (“BQ 123”);(N,N-hexamethylene)-carbamoyl-Leu-D-Trp-(CHO)-D-Trp-OH (“BQ 610”);(R)2-([R-2-[(s)-2-([-hexahydro-1H-azepinyl]-carbonyl]amino-4-methyl-pentanoyl)amino-3-(3[1-methyl-1H-indodyl])propionylamino-3(2-pyridyl) propionicacid (“FR 139317”); cyclo(D-Asp-Pro-D-Ile-Leu-D-Trp) (“JKC 301”);cyclo(D-Ser-Pro-D-Val-Leu-D-Trp) (“JK 302”);5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulphonamide(“BMS 182874”); andN-[1-Formyl-N-[N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl]-D-tryptophan(“BQ 610”). Concentrations for a representative three of these agents isset forth in Table 19.

TABLE 19 Therapeutic and Preferred Concentrations of Spasm InhibitoryAgents Therapeutic Preferred Concentrations Concentrations Class ofAgent (Nanomolar) (Nanomolar) Endothelin Receptor Antagonists: BQ 1230.01-1,000 10-1,000 FR 139317    1-100,000 100-10,000 BQ 610  0.01 to10,000 10-1,000

4. Ca²⁺ Channel Antagonists

Calcium channel antagonists are a distinct group of drugs that interferewith the transmembrane flux of calcium ions required for activation ofcellular responses mediating neuroinflammation. Calcium entry intoplatelets and white blood cells is a key event mediating activation ofresponses in these cells. Furthermore, the role of bradykinin receptorsand neurokinin receptors (NK₁ and NK₂) in mediating theneuroinflammation signal transduction pathway includes increases inintracellular calcium, thus leading to activation of calcium channels onthe plasma membrane. In many tissues, calcium channel antagonists, suchas nifedipine, can reduce the release of arachidonic acid,prostaglandins, and leukotrienes that are evoked by various stimuli.Moncada, S., Flower, R. and Vane, J. in Goodman's and Gilman'sPharmacological Basis of Therapeutics, (7th ed.), MacMillan Publ. Inc.,pp. 660-5 (1995).

Calcium channel antagonists also interfere with the transmembrane fluxof calcium ions required by vascular smooth muscle for contractions.This effect provides the rationale for the use of calcium channelantagonists perioperatively during procedures in which the goal is toalleviate vasospasm and promote relaxation 5 of smooth muscle. Thedihydropyridines, including nisoldipine, act as specific inhibitors(antagonists) of the voltage-dependent gating of the L-type subtype ofcalcium channels. Systemic administration of the calcium channelantagonist nifedipine during cardiac surgery previously has beenutilized to prevent or minimize coronary artery vasospasm. Seitelberger,R., et. al., Circulation, Vol. 83, pp. 460-468 (1991).

Calcium channel antagonists, which are among the anti-spasm agentsuseful in the present invention, exhibit synergistic effect whencombined with other agents of the present invention. Calcium (Ca²⁺)channel antagonists and nitric oxide (NO) donors interact in achievingvasorelaxation or smooth muscle relaxation, i.e., in inhibiting spasmactivity. A rationale for dual use is based upon the fact that these twoclasses of drugs have different molecular mechanisms of action, may notbe completely effective in achieving relaxation used alone, and may havedifferent time periods of effectiveness. In fact, there are numerousstudies showing that calcium channel antagonists alone cannot achievecomplete relaxation of vascular muscle that has been precontracted witha receptor agonist.

The effect of nisoldipine, used alone and in combination withnitroglycerin, on spasm of the internal mammary artery (IMA) showed thatthe combination of the two drugs produced a large positive synergisticeffect in the prevention of contraction (Liu et al., 1994). Thesestudies provide a scientific basis for combination of a calcium channelantagonist and nitric oxide (NO) donor for the efficacious prevention ofvasospasm and relaxation of smooth muscle. Examples of systemicadministration of nitroglycerin and nifedipine during cardiac surgery toprevent and treat myocardial ischemia or coronary artery vasospasm havebeen reported (Cohen et al., 1983; Seitelberger et al., 1991).

Calcium channel antagonists also exhibit synergistic effect withendothelin receptor subtype A (ET_(A)) antagonists. Yanagisawa andcoworkers observed that dihydropyridine antagonists blocked effects ofET-1, an endogenous agonist at the ET_(A) receptor in coronary arterialsmooth muscle, and hence speculated that ET-1 is an endogenous agonistof voltage-sensitive calcium channels. It has been found that thesustained phase of intracellular calcium elevation in smooth musclecells induced by ET_(A) receptor activation requires extracellularcalcium and is at least partially blocked by nicardipine. Thus, theinclusion of a calcium channel antagonist would be expected tosynergistically enhance the actions of an ET_(A) antagonist whencombined in a surgical solution.

Calcium channel antagonists and ATP-sensitive potassium channel openerslikewise exhibit synergistic action. Potassium channels that areATP-sensitive (K_(ATP)) couple the membrane potential of a cell to thecell's metabolic state via sensitivity to adenosine nucleotides. K_(ATP)channels are inhibited by intracellular ATP but are stimulated byintracellular nucleotide diphosphates. The activity of these channels iscontrolled by the electrochemical driving force to potassium andintracellular signals (e.g., ATP or a G-protein), but are not gated bythe membrane potential per se. K_(ATP) channels hyperpolarize themembrane and thus allow them to control the resting potential of thecell. ATP-sensitive potassium currents have been discovered in skeletalmuscle, brain, and vascular and nonvascular smooth muscle. Bindingstudies with radiolabeled ligands have confirmed the existence ofATP-sensitive potassium channels which are the receptor targets for thepotassium-channel opener drugs such as pinacidil. Opening of thesechannels causes potassium efflux and hyperpolarizes the cell membrane.This hyperpolarization (1) induces a reduction in intracellular freecalcium through inhibition of voltage-dependent Ca²⁺ channels byreducing the probability of opening L-type or T-type calcium channels,(2) restrains agonist induced (at receptor operated channels) Ca²⁺release from intracellular sources through inhibition of inositoltriphosphate (IP₃) formation, and (3) lowers the efficiency of calciumas an activator of contractile proteins. The combined actions of thesetwo classes of drugs (ATP-sensitive potassium channel openers andcalcium channel antagonists) will clamp the target cells into a relaxedstate or one which is more resistant to activation.

Finally, calcium channel antagonists and tachykinin and bradykininantagonists exhibit synergistic effects in mediating neuroinflammation.The role of neurokinin receptors in mediating neuroinflammation has beenestablished. The neurokinin₁ (NK₁) and neurokinin₂ (NK₂) receptor(members of the G-protein coupled superfamily) signal transductionpathway includes increases in intracellular calcium, thus leading toactivation of calcium channels on the plasma membrane. Similarly,activation of bradykinin₂ (BK₂) receptors is coupled to increases inintracellular calcium. Thus, calcium channel antagonists interfere witha common mechanism involving elevation of intracellular calcium, part ofwhich enters through L-type channels. This is the basis for synergisticinteraction between calcium channel antagonists and antagonists toneurokinin and bradykinin₂ receptors.

Suitable calcium channel antagonists for the practice of the presentinvention include nisoldipine, nifedipine, nimodipine, lacidipine,isradipine and amlodipine. Suitable concentrations for these agents areset forth in Table 20.

TABLE 20 Therapeutic and Preferred Concentrations of Spasm InhibitoryAgents Therapeutic Preferred Concentrations Concentrations Class ofAgent (Nanomolar) (Nanomolar) Calcium Channel Antagonists: nisoldipine1-10,000 100-1,000 nifedipine 1-10,000 100-5,000 nimodipine 1-10,000100-5,000 lacidipine 1-10,000 100-5,000 isradipine 1-10,000 100-5,000amlodipine 1-10,000 100-5,000

J. Anti-Restenosis Agents

Solutions of the present invention utilized for cardiovascular andgeneral vascular procedures may optionally also include ananti-restenosis agent, particularly for angioplasty, rotationalatherectomy and other interventional vascular uses. The following drugsare suitable for inclusion in the previously described cardiovascularand general vascular irrigation solutions when limitation of restenosisis indicated. The following anti-restenosis agents would preferably becombined with anti-spasm, and still more preferably also withanti-pain/anti-inflammation agents in the solutions of the presentinvention.

1. Antiplatelet Agents

At sites of arterial injury, platelets adhere to collagen and fibrinogenvia specific cell surface receptors, and are then activated by severalindependent mediators. A variety of agonists are able to activateplatelets, including collagen, ADP, thromboxane A2, epinephrine andthrombin. Collagen and thrombin serve as primary activators at sites ofvascular injury, while ADP and thromboxane A2 act to recruit additionalplatelets into a growing platelet plug. The activated plateletsdegranulate and release other agents which serve as chemoattractants andvasoconstrictors, thus promoting vasospasm and platelet accumulation.Thus, anti-platelet agents can be antagonists drawn from any of theabove agonist-receptor targets.

Since platelets play such an important role in the coagulation cascade,oral antiplatelet agents have been routinely administered to patientsundergoing vascular procedures. Indeed, because of this multiplicity ofactivators and observations that single antiplatelet agents are noteffective, some investigators have concluded that a combined treatmentprotocol is necessary for effectiveness. Recently, Willerson andcoworkers reported the intravenous use of 3 combined agents, ridogrel(an antagonist of thromboxane A2), ketanserin (a serotonin antagonist)and clopidogrel (an ADP antagonist). They found that the combination of3 antagonists inhibited several relevant platelet functions and reducedneointimal proliferation in a canine coronary angioplasty model (JACCAbstracts, Feb. 1995). It is still uncertain which approach to treatmentof coronary thrombosis will be most successful. One possibility would beto include an antiplatelet agent and an antithrombotic agent in thecardiovascular and general vascular solutions of the present invention.

a. Thrombin Inhibitors and Receptor Antagonists

Thrombin plays a central role in vascular lesion formation and isconsidered the principal mediator of thrombogenesis. Thus, thrombusformation at vascular lesion sites during and after PTCA (percutaneoustransluminal coronary angioplasty) or other vascular procedure iscentral to acute reocclusion and chronic restenosis. This process can beinterrupted by application of direct anti-thrombins, including hirudinand its synthetic peptide analogs, as well as thrombin receptorantagonist peptides (Harker, et al., 1995, Am. J. Cardiol 75, 12B).Thrombin is also a potent growth factor which initiates smooth musclecell proliferation at sites of vascular injury. In addition, thrombinalso plays a role in modulating the effects of other growth factors suchas PDGF (platelet-derived growth factor), and it has been shown thatthrombin inhibitors reduce expression of PDGF mRNA subsequent tovascular injury induced by balloon angioplasty.

Hirudin is the prototypic direct antithrombin drug since it binds to thecatalytic site and the substrate recognition site (exosite) of thrombin.Animal studies using baboons have shown that this proliferative responsecan be reduced 80% using recombinant hirudin (Ciba-Geigy). Hirulog(Biogen) is a dodecapeptide modeled after hirudin, and binds to theactive site of thrombin via a Phe-Pro-Arg linker molecule. Largeclinical trials of hirudin and hirulog are underway to test theirefficacy in reducing vascular lesions after PTCA and Phase II data onthese inhibitors to date is positive, and both drugs are believed to besuitable in the solutions of the present invention. Preliminary resultsof a 1,200 patient trial with repeat angiographic assessment at 6 monthsto detect restenosis indicated superior shortterm suppression ofischemic events with hirudin vs. heparin. An advantage of this approachis that no significant bleeding complications were reported. Asustained-release local hirulog therapy was found to decrease earlythrombosis but not neointimal thickening after arterial stenting inpigs. Muller, D. et al., Sustained-Release Local Hirulog TherapyDecreases Early Thrombosis but not Neointimal Thickening After ArterialStenting, Am. Heart J. 133, No. 2, pp. 211-218, (1996). In this study,hirulog was released from an impregnated polymer placed around theartery.

Other active anti-thrombin agents being tested which are theorized to besuitable for the present invention are argatroban (Texas Biotechnology)and efegatran (Lilly).

TABLE 21 Therapeutic and Preferred Concentrations of RestenosisInhibitory Agents Therapeutic/Preferred More Concentrations preferredClass of Agent (Nanomolar) (Nanomolar) Thrombin Inhibitors and ReceptorAngtagonists: hirudin 0.00003-3/0.0003-0.3 0.03 hirulog0.2-20,000/2-2,000 200

b. ADP Receptor Antagonists (Purinoceptor Antagonists)

Ticlopidine, an analog of ADP, inhibits both thromboxane and ADP-inducedplatelet aggregation. It is likely that ticlopidine blocks interactionof ADP with its receptor, thereby inhibiting signal transduction by thisG-protein coupled receptor on the surface of platelet membranes. Apreliminary study showed it to be more effective than aspirin incombination with dipyridamole. However, the clinical use of ticlopidinehas been limited because it causes neutropenia. Clopidogrel, aticlopidine analog, is thought to have fewer adverse side effects thanticlopidine and is currently being studied for prevention of ischemicevents. It is theorized that these agents may be suitable for use in thesolutions of the present invention.

c. Thromboxane Inhibitors and Receptor Antagonists

Agents currently utilized for conventional methods of treatment ofthrombosis rely upon aspirin, heparin and plasminogen activators.Aspirin irreversibly acetylates cycloxygenase and inhibits the synthesisof thromboxane A2 and prostacyclin. While data support a benefit ofaspirin for PTCA, the underlying efficacy of aspirin is considered asonly partial or modest. This is likely due to platelet activationthrough thromboxane A2 independent pathways that are not blocked byaspirin induced acetylation of cyclooxygenase. Platelet aggregation andthrombosis may occur despite aspirin treatment. Aspirin in combinationwith dipyridamole has also been shown to reduce the incidence of acutecomplication during PTCA but not the incidence of restenosis.

Two thromboxane receptor antagonists appear to be more efficacious thanaspirin and are believed suitable for use in the solutions and methodsof the present invention. Ticlopidine inhibits both thromboxane andADP-induced platelet aggregation. Ridogrel (R68060) is a combinedthromboxane B2 synthetase inhibitor and thromboxane-prostaglandinendoperoxide receptor blocker. It has been compared with salicylatetherapy in an open-pilot study of patients undergoing PTCA administeredin combination with heparin. Timmermans, C., et al., Ridogrel in theSetting of Percutaneous Transluminal Coronary Angioplasty, Am. J.Cardiol. 68, pp. 463-466, (1991). Treatment consisted of administering aslow intravenous injection of 300 mg just prior to the start of the PTCAprocedure and continued orally after 12 hrs with a dose of 300 mg/twicedaily. From this study, ridogrel was found to be primarily successfulsince no early acute reocclusion occurred in 30 patients. Bleedingcomplications did occur in a significant number (34%) of patients, andthis appears to be a complicating factor that would require specialcare. The study confirmed that ridogrel is a potent long-lastinginhibitor of thromboxane B2 synthetase.

2. Inhibitors of Cell Adhesion Molecules

a. Selectin Inhibitors

Selectin inhibitors block the interaction of a selectin with its cognateligand or receptor. Representative examples of selectin targets at whichthese inhibitors would act include, but are not limited to, E-selectinand P-selectin receptors. Upjohn Co. has licensed rights to a monoclonalantibody developed by Cytel Corps that inhibits the activity ofP-selectin. The product, CY 1748, is in preclinical development, with apotential indication being restenosis.

b. Integrin inhibitors

The platelet glycoprotein IIb/IIIa complex is present on the surface ofresting as well as activated platelets. It appears to undergo atransformation during platelet activation which enables it to serve as abinding site for fibrinogen and other adhesive proteins. Most promisingnew antiplatelet agents are directed at this integrin cell surfacereceptor which represents a final common pathway for plateletaggregation.

Several types of agents fit into the class of GPIIb/IIIa integrinantagonists. A monoclonal antibody, c7E3, (CentoRx; Centocor, MalvernPa.) has been intensively studied to date in a 3,000 patient PTCA study.It is a chimeric human/murine hybrid. A 0.25 mg/kg bolus of c7E3followed by 10 μg/min intravenous infusion for 12 hrs produced greaterthan 80% blockade of GPIIb/IIIa receptors for the duration of theinfusion. This was correlated with a greater than 80% inhibition ofplatelet aggregation. The antibody was coadministered with heparin andan increased risk of bleeding was noted. Additional information wasobtained from the EPIC trial which showed a significant reduction in theprimary end point, a composite of death rate, incidence of nonfatalmyocardial infarction and need for coronary revascularization, andsuggested a long term benefit. Tcheng, (1995) Am. Heart J. 130, 673-679.A phase IV study (EPILOG) designed to address safety and efficacy issueswith c7E3 Fab is planned or in progress. This monoclonal antibody canalso be classified as a platelet membrane glycoprotein receptorantagonist directed against the glycoprotein IIb/IIIa receptor.

The platelet glycoprotein IIba/IIIa receptor blocker, integrelin, is acyclic heptapeptide that is highly specific for this molecular target.In contrast to the antibody, it has a short biologic half-life (about 10minutes). The safety and efficacy of integrelin was first evaluated inthe Phase II Impact trial. Either 4 or 12 hour intravenous infusions of1.0 μg/kg/min of integrelin were utilized (Topol, E., 1995 Am. J.Cardiol, 27B-33B). It was provided in combination with other agents(heparin, aspirin) and was shown to exhibit potent anti-plateletaggregation properties (>80%). A phase III study, the IMPACT II trial,of 4000 patients showed that integrelin markedly reduced ischemic eventsin patients who had undergone Rotablator atherectomy (JACC Abstracts,1996). Suitable concentrations of the drugs c7E3 and integrelin for usein the present invention are set forth below.

In addition, two peptidomimetics, MK-383 (Merck) and RO 4483(Hoffmann-LaRoche), have been studied in Phase II clinicals. Since theseare both small molecules, they have a short half-life and high potency.However, these seem to also have less specificity, interacting withother closely related integrins. It is theorized that thesepeptidomimetics may also be suitable for use in the present invention.

TABLE 22 Therapeutic and Preferred Concentrations of RestenosisInhibitory Agents Therapeutic/Preferred More Concentrations preferredClass of Agent (Nanomolar) (Nanomolar) Cell Adhesion Inhibitors: c7E30.5-50,000/5-5,000 500 Integrelin 0.1-10,000/1-1000 × K_(d) 100 × K_(d)

3. Anti-chemotactic agents

Anti-chemotactic agents prevent the chemotaxis of inflammatory cells.Representative examples of anti-chemotactic targets at which theseagents would act include, but are not limited to, F-Met-Leu-Phereceptors, IL-8 receptors, MCP-1 receptors, and MIP-1-α/RANTESreceptors. Drugs within this class of agents are early in thedevelopment stage, but it is theorized that they may be suitable for usein the present invention.

4. Interleukin Receptor Antagonists

Interleukin receptor antagonists are agents which block the interactionof an interleukin with its cognate ligand or receptor. Specific receptorantagonists for any of the numerous interleukin receptors are early inthe development process. The exception to this is the naturallyoccurring existence of a secreted form of the IL-1 receptor, referred toas IL-1 antagonist protein (IL-1AP). This antagonist binds IL-1 and hasbeen shown to suppress the biological actions of IL-1, and is theorizedto be suitable for the practice of the present invention.

5. Intracellular Signaling Inhibitors

a. Protein Kinase Inhibitors

i. Protein Kinase C (PKC) Inhibitors

Protein kinase C (PKC) plays a crucial role in cell-surface signaltransduction for a number of physiological processes. PKC isozymes canbe activated as downstream targets resulting from initial activation ofeither G-protein coupled receptors (e.g., serotonin, endothelin, etc.)or growth-factor receptors such as PDGF. Both of these receptor classesplay important roles in mediating vascular spasm and restenosissubsequent to coronary balloon angioplasty procedures.

Molecular cloning analysis has revealed that PKC exists as a largefamily consisting of at least 8 subspecies (isozymes). These isozymesdiffer substantially in structure and mechanism for linking receptoractivation to changes in the proliferative response of specific cells.Expression of specific isozymes is found in a wide variety of celltypes, including: platelets, neutrophils, myeloid cells, and smoothmuscle cells. Inhibitors of PKC are therefore likely to effect signalingpathways in several cell types unless the inhibitor shows isozymespecificity. Thus, inhibitors of PKC can be predicted to be effective inblocking the proliferative response of smooth muscle cells and may alsohave an anti-inflammatory effect in blocking neutrophil activation andsubsequent attachment. Several inhibitors have been described andinitial reports indicate an IC₅₀ of 50 nM for calphostin C inhibitoryactivity. G-6203 (also known as Go 6976) is a new, potent PKC inhibitorwith high selectivity for certain PKC isotypes with IC₅₀ values in the2-10 nM range. Concentrations of these and another drug, GF 109203X,also known as Go 6850 or bisindoylmaleimide I (available fromWarner-Lambert), that are believed to be suitable for use in the presentinvention are set forth below.

TABLE 23 Therapeutic and Preferred Concentrations of RestenosisInhibitory Agents Therapeutic/Preferred More Concentrations preferredClass of Agent (Nanomolar) (Nanomolar) Protein Kinase C Inhibitors:calphostin C 0.5-50,000/100-5,000 500 GF 109203X 0.1-10,000/1-1,000  100G-6203 (Go 6976) 0.1-10,000/1-1,000  100

ii. Protein tyrosine kinase inhibitors

Although there is a tremendous diversity among the numerous members ofthe receptors tyrosine-kinase (RTK) family, the signaling mechanismsused by these receptors share many common features. Biochemical andmolecular genetic studies have shown that binding of the ligand to theextracellular domain of the RTK rapidly activates the intrinsic tyrosinekinase catalytic activity of the intracellular domain (see FIG. 5). Theincreased activity results in tyrosine-specific phosphorylation of anumber of intracellular substrates which contain a common sequencemotif. Consequently, this causes activation of numerous “downstream”signaling molecules and a cascade of intracellular pathways thatregulate phospholipid metabolism, arachidonate metabolism, proteinphosphorylation (involving mechanisms other than protein kinases),calcium mobilization and transcriptional activation (see FIG. 2).Growth-factor-dependent tyrosine kinase activity of the RTK cytoplasmicdomain is the primary mechanism for generation of intracellular signalsthat lead to cellular proliferation. Thus, inhibitors have the potentialto block this signaling and thereby prevent the proliferative response(see FIG. 5).

The platelet-derived growth factor (PDGF) receptor is of great interestas a target for inhibition in the cardiovascular field since it isbelieved to play a significant role both in atherosclerosis andrestenosis. The release of PDGF by platelets at damaged surfaces ofendothelium within blood vessels results in stimulation of PDGFreceptors on vascular smooth muscle cells. As described above, thisinitiates a sequence of intracellular events leading to enhancedproliferation and neointimal thickening. An inhibitor of PDGF kinaseactivity would be expected to prevent proliferation and enhance theprobability of success following cardiovascular and general vascularprocedures. Any of several related tyrphostin compounds have potentialas specific inhibitors of PDGF-receptor tyrosine kinase activity (IC₅₀sin vitro in the 0.5-1.0 μM range), since they have little effect onother protein kinases and other signal transduction systems. To date,only a few of the many tyrphostin compounds are commercially available,and suitable concentrations for these agents as used in the presentinvention are set forth below. In addition, staurosporine has beenreported to demonstrate potent inhibitory effects against severalprotein tyrosine kinases of the src subfamily and a suitableconcentration for this agent as used in the present invention also isset forth below.

TABLE 24 Therapeutic and Preferred Concentrations of RestenosisInhibitory Agents Therapeutic/Preferred More Concentrations preferredClass of Agent (Nanomolar) (Nanomolar) Protein Kinase Inhibitorslavendustin A 10-100,000/100-10,000 10,000 tyrphostin10-100,000/100-20,000 10,000 AG1296 tyrphostin 10-100,000/100-20,00010,000 AG1295 staurosporine 1-100,000/10-10,000  1,000

b. Modulators of Intracellular Protein Tyrosine Phosphatases.

Non-transmembrane protein tyrosine phosphatases (PTPases) containingsrc-homology₂ SH2 domains are known and nomenclature refers to them asSH-PTP1 and SH-PTP₂. In addition, SH-PTP1 is also known as PTP₁C, HCP orSHP. SH-PTP₂ is also known as PTP₁D or PTP₂C. Similarly, SH-PTP₁ isexpressed at high levels in hematopoietic cells of all lineages and allstages of differentiation, and the SH-PTP₁ gene has been identified asresponsible for the motheaten (me) mouse phenotype and this provides abasis for predicting the effects of inhibitors that would block itsinteraction with its cellular substrates. Stimulation of neutrophilswith chemotactic peptides is known to result in the activation oftyrosine kinases that mediate neutrophil responses (Cui, et al., 1994 J.Immunol.) and PTPase activity modulates agonist induced activity byreversing the effects of tyrosine kinases activated in the initialphases of cell stimulation. Agents that could stimulate PTPase activitycould have potential therapeutic applications as anti-inflammatorymediators.

These same PTPases have also been shown to modulate the activity ofcertain RTKs. They appear to counter-balance the effect of activatedreceptor kinases and thus may represent important drug targets. In vitroexperiments show that injection of PTPase blocks insulin stimulatedphosphorylation of tyrosyl residues on endogenous proteins. Thus,activators of PTPase activity could serve to reverse activation ofPDGF-receptor action in restenosis, and are believed to be useful in thesolutions of the present invention. In addition, receptor-linked PTPasesalso function as extracellular ligands, similar to those of celladhesion molecules. The functional consequences of the binding of aligand to the extracellular domain have not yet been defined but it isreasonable to assume that binding would serve to modulate phosphataseactivity within cells (Fashena and Zinn, 1995, Current Biology, 5,1367-1369) . Such actions could block adhesion mediated by other cellsurface adhesion molecules (NCAM) and provide an anti-inflammatoryeffect. No drugs have been developed yet for these applications.

c. Inhibitors of SH2 Domains (src Homology₂ Domains).

SH2 domains, originally identified in the src subfamily of proteintyrosine kinases (PTKs), are noncatalytic protein sequences and consistof about 100 amino acids conserved among a variety of signal transducingproteins (Cohen, et al., 1995). SH2 domains function asphosphotyrosine-binding modules and thereby mediate criticalprotein-protein associations in signal transduction pathways withincells (Pawson, Nature, 573-580, 1995). In particular, the role of SH2domains has been clearly defined as critical for receptor tyrosinekinase (RTK) mediated signaling such as in the case of theplatelet-derived growth factor (PDGF) receptor.Phosphotyrosine-containing sites on autophosphorylated RTKs serve asbinding sites for SH2-proteins and thereby mediate the activation ofbiochemical signaling pathways (see FIG. 2) (Carpenter, G., FASEB J.6:3283-3289, 1992; Sierke, S. and Koland, J. Biochem. 32:10102-10108,1993). The SH2 domains are responsible for coupling the activatedgrowth-factor receptors to cellular responses which include alterationsin gene expression, and ultimately cellular proliferation (see FIG. 5).Thus, inhibitors that will selectively block the effects of activationof specific RTKs expressed on the surface of vascular smooth musclecells are predicted to be effective in blocking proliferation and therestenosis process after PTCA or other vascular procedure. One RTKtarget of current interest is the PDGF receptor.

At least 20 cytosolic proteins have been identified that contain SH2domains and function in intracellular signaling. The distribution of SH2domains is not restricted to a particular protein family, but found inseveral classes of proteins, protein kinases, lipid kinases, proteinphosphatases, phospholipases, Ras-controlling proteins and sometranscription factors. Many of the SH2-containing proteins have knownenzymatic activities while others (Grb2 and Crk) function as “linkers”and “adapters” between cell surface receptors and “downstream” effectormolecules (Marengere, L., et al., Nature 369:502-505, 1994). Examples ofproteins containing SH2 domains with enzymatic activities that areactivated in signal transduction include, but are not limited to, thesrc subfamily of protein tyrosine kinases (src (pp60^(c-src)), abl, lck,fyn, fgr and others), phospholipaseCγ (PLCγ), phosphatidylinositol3-kinase (PI-3-kinase), p21-ras GTPase activating protein (GAP) and SH2containing protein tyrosine phosphatases (SH-PTPases) (Songyang, et al.,Cell 72, 767-778, 1993). Due to the central role these variousSH2-proteins occupy in transmitting signals from activated cell surfacereceptors into a cascade of additional molecular interactions thatultimately define cellular responses, inhibitors which block specificSH2 protein binding are desirable as agents for a variety of potentialtherapeutic applications.

In addition, the regulation of many immune/inflammatory responses ismediated through receptors that transmit signals through non-receptortyrosine kinases containing SH2 domains. T-cell activation via theantigen specific T-cell receptor (TCR) initiates a signal transductioncascade leading to lymphokine secretion and T-cell proliferation. One ofthe earliest biochemical responses following TCR activation is anincrease in tyrosine kinase activity. In particular, neutrophilactivation is in part controlled through responses of the cell surfaceimmunoglobulin G receptors. Activation of these receptors mediatesactivation of unidentified tyrosine kinases which are known to possessSH2 domains. Additional evidence indicates that several src-familykinases (Ick, blk, fyn) participate in signal transduction pathwaysleading from cytokine and integrin receptors and hence may serve tointegrate stimuli received from several independent receptor structures.Thus, inhibitors of specific SH2 domains have the potential to blockmany neutrophil functions and serve as anti-inflammatory mediators.

Efforts to develop drugs targeted to SH2 domains currently are beingconducted at the biochemical in vitro and cellular level. Should suchefforts be successful, it is theorized that the resulting drugs would beuseful in the practice of the present invention.

d. Calcium Channel Antagonists

Calcium channel antagonists, previously described with relation to spasminhibitory function, also can be used as anti-restenotic agents in thecardiovascular and general vascular solutions of the present invention.Activation of growth factor receptors, such as PDGF, is known to resultin an increase in intracellular calcium (see FIG. 2). Studies at thecellular level have shown that actions of calcium channel antagonistsare effective at inhibiting mitogenesis of vascular smooth muscle cells.

6. Synergistic Interactions Derived From Therapeutic Combinations OfAnti-Restenosis Agents And Other Agents Used In Cardiovascular andGeneral Vascular Solutions

Given the complexity of the disease process associated with restenosisafter PTCA or other cardiovascular or general vascular therapeuticprocedure and the multiplicity of molecular targets involved, blockadeor inhibition of a single molecular target is unlikely to provideadequate efficacy in preventing vasospasm and restenosis (see FIG. 2).Indeed, a number of animal studies targeting different individualmolecular receptors and or enzymes have not proven effective in animalmodels or have not yielded efficacy for both pathologies in clinicaltrials to date. (Freed, M., et al., An Intensive Poly-pharmaceuticalApproach to the Prevention of Restenosis: the Mevacor, Ace Inhibitor,Colchicine (BIG-MAC) Pilot Trial, J. Am. Coll. of Cardiol. 21, p. 33A,(1993). Serruys, P., et al., PARK: the Post Angioplasty RestenosisKetanserin Trial, J. Am. Coll. of Cardiol. 21, p. 322A, (1993).Therefore, a therapeutic combination of drugs acting on distinctmolecular targets and delivered locally appears necessary for clinicaleffectiveness in the therapeutic approach to vasospasm and restenosis.As described below, the rationale for this synergistic moleculartargeted therapy is derived from recent advances in understandingfundamental biochemical mechanisms by which vascular smooth muscle cellsin the vessel wall transmit and integrate stimuli to which they areexposed during PTCA or other vascular interventional procedure.

a. “Crosstalk” and Convergence in Major Signaling Pathways

The molecular switches responsible for cell signaling have beentraditionally divided into two major discrete signaling pathways, eachcomprising a distinct set of protein families that act as transducersfor a particular set of extracellular stimuli and mediating distinctcell responses. One such pathway transduces signals fromneurotransmitters and hormones through G-protein coupled receptors(GPCRs) to produce contractile responses using intracellular targets oftrimeric G proteins and Ca²⁺ (see FIG. 2). These stimuli and theirrespective receptors mediate smooth muscle contraction and may inducevasospasm in the context of PTCA or other cardiovascular or generalvascular therapeutic or diagnostic procedure. Examples of signalingmolecules involved in mediating spasm through the GPCR pathway are 5-HTand endothelin for which antagonists have been included acting via theirrespective G-protein coupled receptors.

A second major pathway transduces signals from growth factors, such asPDGF, through tyrosine kinases, adaptor proteins and the Ras proteininto regulation of cell proliferation and differentiation (see FIGS. 2and 5). This pathway may also be activated during PTCA or othercardiovascular or general vascular procedure leading to a high incidenceof vascular smooth muscle cell proliferation. An example of a restenosisdrug target is the PDGF-receptor.

Signals transmitted from neurotransmitters and hormones stimulate eitherof two classes of receptors: G-protein-coupled receptors, composed ofseven-helix transmembrane regions, or ligand-gated ion channels.“Downstream” signals from both kinds of receptors converge oncontrolling the concentration of cytoplasmic Ca²⁺ which triggerscontraction in smooth muscle cells (see FIG. 2). Each GPCR transmembranereceptor activates a specific class of trimeric G proteins, includingG_(q), G_(i) or many others. Gα and/or G_(βγ) subunits activatephospholipase C_(γ), resulting in activation of protein kinase C (PKC)and an increase in the levels of cytoplasmic calcium by release ofcalcium from intracellular stores.

Growth factor signaling, such as mediated by PDGF, converges onregulation of cell growth. This pathway depends upon phosphorylation oftyrosine residues in receptor tyrosine kinases and “downstream” enzymes(phospholipase C_(γ), discussed above with regard to tyrosine kinases).Activation of the PDGF-receptor also leads to stimulation of PKC andelevation of intracellular calcium, common steps shared by the GPCRs(see FIG. 2). It is now recognized that ligand-independent “crosstalk”can transactivate tyrosine kinase receptor pathways in response tostimulation of GPCRs. Recent work has identified Shc, an adaptor proteinin the tyrosine kinase/Ras pathway, as a key intermediary protein thatrelays messages from the GPCR pathway described above to the tyrosinekinase pathway (see FIG. 2) (Lev et al., 1995, Nature 376:737).Activation of Shc is calcium dependent. Thus, a combination of selectiveinhibitors which blocks transactivation of a common signaling pathwayleading to vascular smooth muscle cell proliferation will actsynergistically to prevent spasm and restenosis after PTCA or othercardiovascular or general vascular procedure. Specific examples arebriefly detailed below.

b. Synergistic Interactions between PKC Inhibitors and Calcium ChannelAntagonists

In this case synergistic interactions among PKC inhibitors and calciumchannel antagonists in achieving vasorelaxation and inhibition ofproliferation occur due to “crosstalk” between GPCR and tyrosine kinasesignaling pathways (see FIG. 2). A rationale for dual use is based uponthe fact that these drugs have different molecular mechanisms of action.As described above, GPCR stimulation results in activation of proteinkinase C and an increase in the levels of cytoplasmic calcium by releaseof calcium from intracellular stores. Calcium-activated PKC is a centralcontrol point in the transmission of extracellular responses.“Crosstalk” from GPCR stimulated pathways through PKC can lead tomitogenesis of vascular smooth muscle cells and thus calcium channelantagonists will have the dual action of directly blocking spasm andfurther preventing activation of proliferation by inhibiting Shcactivation. Conversely, the PKC inhibitor acts on part of the pathwayleading to contraction.

c. Synergistic Effects of PKC Inhibitors, 5-HT₂ Antagonists and ET_(A)Antagonists

The 5-HT₂ receptor family contains three members designated 5-HT_(2A),5-HT_(2B), and 5-HT_(2C), all of which share the common property ofbeing coupled to phosphotidylinositol turnover and increases inintracellular calcium (Hoyer et al., 1988, Hartig et al., 1989). Thedistribution of these receptors includes vascular smooth muscle andplatelets and, due to their localization, these 5-HT receptors areimportant in mediating spasm, thrombosis and restenosis. It has beenfound that the sustained phase of intracellular calcium elevation insmooth muscle cells induced by ET_(A) receptor activation requiresextracellular calcium and is at least partially blocked by nicardipine.Since activation of both 5-HT₂ receptors and ET_(A) receptors ismediated through calcium, the inclusion of a PKC inhibitor is expectedto synergistically enhance the actions of antagonists to both of thesereceptors when combined in a surgical solution (see FIGS. 2 and 4).

d. Synergistic Effects of Protein Tyrosine Kinase Inhibitors and CalciumChannel Antagonists

The mitogenic effect of PDGF (or basic fibroblast growth factor orinsulin-like-growth-factor-1) is mediated through receptors that possessintrinsic protein tyrosine kinase activity. The substrates for PDGFphosphorylation are many and lead to activation of mitogen-activatedprotein kinases (MAPK) and ultimately proliferation (see FIG. 5). Theendothelin, 5-HT and thrombin receptors, which are members of theG-protein coupled superfamily, trigger a signal transduction pathwaywhich includes increases in intracellular calcium, leading to activationof calcium channels on the plasma membrane. Thus, calcium channelantagonists interfere with a common mechanism employed by these GPCRs.It has recently been shown that activation of certain GPCRs, includingendothelin and bradykinin, leads to a rapid increase in tyrosinephosphorylation of a number of intracellular proteins. Some of theproteins phosphorylated parallel those known necessary for mitogenicstimulation. The rapidity of the process was such that changes weredetectable in seconds and the targets acted upon likely play a role inmitogenesis. These tyrosine phosphorylation events were not blocked by aselective PKC inhibitor or apparently mediated by increasedintracellular calcium. Thus, since two independent pathways, the GPCRand tyrosine phosphorylation pathways, can drive the vascular smoothmuscle cells into a proliferative state, it is necessary to block bothindependent signaling arms. This is the basis for the synergisticinteraction between calcium channel antagonists and tyrosine kinaseinhibitors in the surgical solution. Because the actions of the proteintyrosine kinase inhibitors in preventing vascular smooth muscle cellproliferation occur via independent molecular pathways (described above)from those involving calcium and protein kinase C, the combination ofthe two classes of drugs, calcium channel antagonists and proteintyrosine kinase inhibitors, is expected to be more efficacious ininhibiting spasm and restenosis than employing either single class ofdrug alone.

e. Synergistic Effects of Protein Tyrosine Kinase Inhibitors andThrombin Receptor Antagonists

Thrombin mediates its action via the thrombin receptor, another memberof the GPCR superfamily. Binding to the receptor stimulates plateletaggregation, smooth muscle cell contraction and mitogenesis. Signaltransduction occurs through multiple pathways: activation ofphospholipse (PLC) through Gproteins and activation of tyrosine kinases.The activation of tyrosine kinase activity is also essential formitogenesis of the vascular smooth muscle cells. Experiments have shownthat inhibition with a specific tyrosine kinase inhibitor was effectivein blocking thrombin-induced mitosis, although there were no effects onthe PLC pathway as monitored by measurement of intracellular calcium(Weiss and Nucitelli, 1992, J. Biol. Chem. 267:5608-5613). Because theactions of the protein tyrosine kinase inhibitors in preventing vascularsmooth muscle cell proliferation occur via independent molecularpathways (described above) from those involving calcium and proteinkinase C, the combination of protein tyrosine kinase inhibitors andthrombin receptor antagonists is anticipated to be more efficacious ininhibiting platelet aggregation, spasm and restenosis than employingeither class of agent alone.

VI. Method of Application

The solution of the present invention has applications for a variety ofoperative/interventional procedures, including surgical, diagnostic andtherapeutic techniques. The irrigation solution is perioperativelyapplied during arthroscopic surgery of anatomic joints, urologicalprocedures, cardiovascular and general vascular diagnostic andtherapeutic procedures and for general surgery. As used herein, the term“perioperative” encompasses application intraprocedurally, pre- andintraprocedurally, intra- and postprocedurally, and pre-, intra- andpostprocedurally. Preferably the solution is applied preprocedurallyand/or postprocedurally as well as intraprocedurally. Such proceduresconventionally utilize physiologic irrigation fluids, such as normalsaline or lactated Ringer's, applied to the surgical site by techniqueswell known to those of ordinary skill in the art. The method of thepresent invention involves substituting theanti-pain/anti-inflammatory/anti-spasm/anti-restenosis irrigationsolutions of the present invention for conventionally applied irrigationfluids. The irrigation solution is applied to the wound or surgical siteprior to the initiation of the procedure, preferably before tissuetrauma, and continuously throughout the duration of the procedure, topreemptively block pain and inflammation, spasm and restenosis. As usedherein throughout, the term “irrigation” is intended to mean theflushing of a wound or anatomic structure with a stream of liquid. Theterm “application” is intended to encompass irrigation and other methodsof locally introducing the solution of the present invention, such asintroducing a gellable version of the solution to the operative site,with the gelled solution then remaining at the site throughout theprocedure. As used herein throughout, the term “continuously” isintended to also include situations in which there is repeated andfrequent irrigation of wounds at a frequency sufficient to maintain apredetermined therapeutic local concentration of the applied agents, andapplications in which there may be intermittent cessation of irrigationfluid flow necessitated by operating technique.

The concentrations listed for each of the agents within the solutions ofthe present invention are the concentrations of the agents deliveredlocally, in the absence of metabolic transformation, to the operativesite in order to achieve a predetermined level of effect at theoperative site. It is understood that the drug concentrations in a givensolution may need to be adjusted to account for local dilution upondelivery. For example, in the cardiovascular application, if one assumesan average human coronary artery blood flow rate of 80 cc per minute andan average delivery rate for the solution of 5 cc per minute via a localdelivery catheter (i.e., a blood flow-to-solution delivery ratio of 16to 1), one would require that the drug concentrations within thesolution be increased 16-fold over the desired in vivo drugconcentrations. Solution concentrations are not adjusted to account formetabolic transformations or dilution by total body distribution becausethese circumstances are avoided by local delivery, as opposed to oral,intravenous, subcutaneous or intramuscular application.

Arthroscopic techniques for which the present solution may be employedinclude, by way of non-limiting example, partial meniscectomies andligament reconstructions in the knee, shoulder acromioplasties, rotatorcuff debridements, elbow synovectomies, and wrist and anklearthroscopies. The irrigation solution is continuously suppliedintraoperatively to the joint at a flow rate sufficient to distend thejoint capsule, to remove operative debris, and to enable unobstructedintra-articular visualization.

A suitable irrigation solution for control of pain and edema during sucharthroscopic techniques is provided in Example I herein below. Forarthroscopy, it is preferred that the solution include a combination,and preferably all, or any of the following: a serotonin2 receptorantagonist, a serotonin₃ receptor antagonist, a histamine₁ receptorantagonist, a serotonin receptor agonist acting on the 1A, 1B, 1D, 1Fand/or 1E receptors, a bradykinin₁ receptor antagonist, a bradykinin₂receptor antagonist, and a cyclooxygenase inhibitor.

This solution utilizes extremely low doses of these pain andinflammation inhibitors, due to the local application of the agentsdirectly to the operative site during the procedure. For example, lessthan 0.05 mg of amitriptyline (a suitable serotonin₂ and histamine₁“dual” receptor antagonist) are needed per liter of irrigation fluid toprovide the desired effective local tissue concentrations that wouldinhibit 5-HT₂ and H₁ receptors. This dosage is extremely low relative tothe 10-25 mg of oral amitriptyline that is the usual starting dose forthis drug. This same rationale applies to the anti-spasm andanti-restenosis agents which are utilized in the solution of the presentinvention to reduce spasm associated with urologic, cardiovascular andgeneral vascular procedures and to inhibit restenosis associated withcardiovascular and general vascular procedures. For example, less than0.2 mg of nisoldipine (a suitable calcium channel antagonist) isrequired per liter of irrigation fluid to provide the desired effectivelocal tissue concentrations that would inhibit the voltage-dependentgating of the L-subtype of calcium channels. This dose is extremely lowcompared to the single oral dose of nisoldipine which is 20 to 40 mg.

In each of the surgical solutions of the present invention, the agentsare included in low concentrations and are delivered locally in lowdoses relative to concentrations and doses required with conventionalmethods of drug administration to achieve the desired therapeuticeffect. It is impossible to obtain an equivalent therapeutic effect bydelivering similarly dosed agents via other (i.e., intravenous,subcutaneous, intramuscular or oral) routes of drug administration sincedrugs given systemically are subject to first- and second-passmetabolism.

For example, using a rat model of arthroscopy, the inventors examinedthe ability of amitriptyline, a 5-HT₂ antagonist, to inhibit5-HT-induced plasma extravasation in the rat knee in accordance with thepresent invention. This study, described more fully below in ExampleXII, compared the therapeutic dosing of amitriptyline delivered locally(i.e., intra-articularly) at the knee and intravenously. The resultsdemonstrated that intra-articular administration of amitriptylinerequired total dosing levels approximately 200-fold less than wererequired via the intravenous route to obtain the same therapeuticeffect. Given that only a small fraction of the drug deliveredintra-articularly is absorbed by the local synovial tissue, thedifference in plasma drug levels between the two routes ofadministration is much greater than the difference in totalamitriptyline dosing levels.

Practice of the present invention should be distinguished fromconventional intra-articular injections of opiates and/or localanesthetics at the completion of arthroscopic or “open” joint (e.g.,knee, shoulder, etc.) procedures. The solution of the present inventionis used for continuous infusion throughout the surgical procedure toprovide preemptive inhibition of pain and inflammation. In contrast, thehigh concentrations necessary to achieve therapeutic efficacy with aconstant infusion of local anesthetics, such as lidocaine (0.5-2%solutions), would result in profound systemic toxicity.

Upon completion of the procedure of the present invention, it may bedesirable to inject or otherwise apply a higher concentration of thesame pain and inflammation inhibitors as used in the irrigation solutionat the operative site, as an alternative or supplement to opiates.

The solution of the present invention also has application incardiovascular and general vascular diagnostic and therapeuticprocedures to potentially decrease vessel wall spasm, plateletaggregation, vascular smooth muscle cell proliferation and nociceptoractivation produced by vessel manipulation. Reference herein to arterialtreatment is intended to encompass the treatment of venous graftsharvested and placed in the arterial system. A suitable solution forsuch techniques is disclosed in Example II herein below. Thecardiovascular and general vascular solution preferably includes anycombination, and preferably all, of the following: a 5-HT₂ receptorantagonist (Saxena, P. R., et. al., Cardiovascular Effects of SerotoninInhibitory Agonists and Antagonists, J Cardiovasc Pharmacol 15 (Suppl.7), pp. S17-S34 (1990); Douglas, 1985); a 5-HT₃ receptor antagonist toblock activation of these receptors on sympathetic neurons and C-fibernociceptive neurons in the vessel walls, which has been shown to producebrady- and tachycardia (Saxena et. al. 1990);

a bradykinin₁ receptor antagonist; and a cyclooxygenase inhibitor toprevent production of prostaglandins at tissue injury sites and therebydecreasing pain and inflammation. In addition, the cardiovascular andgeneral vascular solution also preferably will contain a serotonin_(1B)(also known as serotonin_(1Dβ)) antagonist because serotonin has beenshown to produce significant vascular spasm via activation of theserotonin_(1B) receptors in humans. Kaumann, A.J., et al., VariableParticipation of 5-HT1-Like Receptors and 5-HT2 Receptors inSerotonin-Induced Contraction of Human Isolated Coronary Arteries,Circulation 90, pp. 1141-53 (1994). This excitatory action ofserotonin_(1B) receptors in vessel walls, resulting in vasoconstriction,is in contrast to the previously-discussed inhibitory action ofserotonin_(1B) receptors in neurons. The cardiovascular and generalvascular solution of the present invention also may suitably include oneor more of the anti-restenosis agents disclosed herein that reduce theincidence and severity of post-procedural restenosis resulting from, forexample, angioplasty or rotational atherectomy.

The solution of the present invention also has utility for reducing painand inflammation associated with urologic procedures, such astrans-urethral prostate resection and similar urologic procedures.References herein to application of solution to the urinary tract or tothe urological structures is intended to include application to theurinary tract per se, bladder and prostate and associated structures.Studies have demonstrated that serotonin, histamine and bradykininproduce inflammation in lower urinary tract tissues. Schwartz, M. M.,et. al., Vascular Leakage in the Kidney and Lower Urinary Tract: Effectsof Histamine, Serotonin and Bradykinin, Proc Soc Exp Biol Med 140, pp.535-539 (1972). A suitable irrigation solution for urologic proceduresis disclosed in Example III herein below. The solution preferablyincludes a combination, and preferably all, of the following: ahistamine₁ receptor antagonist to inhibit histamine-induced pain andinflammation; a 5-HT₃ receptor antagonist to block activation of thesereceptors on peripheral C-fiber nociceptive neurons; a bradykinin₁antagonist; a bradykinin₂ antagonist; and a cyclooxygenase inhibitor todecrease pain/inflammation produced by prostaglandins at the tissueinjury sites. Preferably an anti-spasm agent is also included to preventspasm in the urethral canal and bladder wall.

Some of the solutions of the present invention may suitably also includea gelling agent to produce a dilute gel. This gellable solution may beapplied, for example, within the urinary tract or an arterial vessel todeliver a continuous, dilute local predetermined concentration ofagents.

The solution of the present invention may also be employedperioperatively for the inhibition of pain and inflammation in surgicalwounds, as well as to reduce pain and inflammation associated withburns. Bums result in the release of a significant quantity of biogenicamines, which not only produce pain and inflammation, but also result inprofound plasma extravasation (fluid loss), often a life-threateningcomponent of severe burns. Holliman, C. J., et. al., The Effect ofKetanserin, a Specific Serotonin Antagonist, on Burn Shock HemodynamicParameters in a Porcine Burn Model, J Trauma 23, pp. 867-871 (1983). Thesolution disclosed in Example I for arthroscopy may also be suitablyapplied to a wound or burn for pain and inflammation control, and forsurgical procedures such as arthroscopy. The agents of the solution ofExample I may alternately be included in a paste or salve base, forapplication to the burn or wound.

VII. Examples

The following are several formulations in accordance with the presentinvention suitable for certain operative procedures followed by asummary of three clinical studies utilizing the agents of the presentinvention.

A. Example I Irrigation Solution for Arthroscopy

The following composition is suitable for use in anatomic jointirrigation during arthroscopic procedures. Each drug is solubilized in acarrier fluid containing physiologic electrolytes, such as normal salineor lactated Ringer's solution, as are the remaining solutions describedin subsequent examples.

Concentration Class (Nanomolar): Most of Agent Drug TherapeuticPreferred Preferred serotonin₂ amitriptyline 0.1-1,000 50-500 100antagonist serotonin₃ meto-   10-10,000  200-2,000 1,000   antagonistclopramide histamine₁ amitriptyline 0.1-1,000 50-500 200 antagonistserotonin_(1A,) sumatriptan   1-1,000 10-200  50 _(IB, 1D,) _(1F)agonist bradykinin₁ [des-Arg¹⁰]  1-1,000 50-500 200 agonist derivativeof HOE 140 bradykinin₂ HOE 140  1-1,000 50-500 200 agonist

B. Example II Irrigation Solution for Cardiovascular and GeneralVascular Therapeutic and Diagnostic Procedures

The following drugs and concentration ranges in solution in aphysiologic carrier fluid are suitable for use in irrigating operativesites during cardiovascular and general vascular procedures.

Concentration Class (Nanomolar): Most of Agent Drug TherapeuticPreferred Preferred serotonin₂ trazodone 0.1-2,000 50-500   200antagonist serotonin₃ meto-   10-10,000  200-2,000 1,000 antagonistclopramide serotonin_(1B) yohimbine 0.1-1,000 50-500   200 antagonistbradykinin₁ [des-Arg¹⁰]   1-1,000 50-500   200 antagonist derivative ofHOE 140 cyclo- ketorolac  100-10,000  500-5,000 3,000 oxygenaseinhibitor

C. Example III Irrigation Solution for Urologic Procedures

The following drugs and concentration ranges in solution in aphysiologic carrier fluid are suitable for use in irrigating operativesites during urologic procedures.

Concentration Class (Nanomolar): Most of Agent Drug TherapeuticPreferred Preferred histamine₁ terfenadine 0.1-1,000   50-500   200antagonist serotonin₃ meto- 10-10,000  200-2,000 1,000 antagonistclopramide bradykinin₁ [des-Arg¹⁰] 1-1,000 50-500   200 antagonistderivative of HOE 140 bradykinin₂ HOE 140 1-1,000 50-500   200antagonist cyclo- 100-10,000  500-5,000 3,000 oxygenase inhibitor

D. Example IV Irrigation Solution for Arthroscopy, Burns, GeneralSurgical Wounds and Oral/Dental Applications

The following composition is preferred for use in anatomic irrigationduring arthroscopic and oral/dental procedures and the management ofburns and general surgical wounds. While the solution set forth inExample I is suitable for use with the present invention, the followingsolution is even more preferred because of expected higher efficacy.

Concentration Class (Nanomolar): Most of Agent Drug TherapeuticPreferred Preferred serotonin₂ amitriptyline 0.1-1,000   50-500   200antagonist serotonin₃ meto- 10-10,000  200-2,000 1,000 antagonistclopramide histamine₁ amitriptyline 0.1-1,000   50-500   200 antagonistserotonin_(1A,) sumatriptan 1-1,000 10-200   100 _(1B, 1D,) _(1F)agonist cyclo- ketorolac 100-10,000   500-5,000 3,000 oxygenaseinhibitor neurokinin₁ GR 82334 1-1,000 10-500   200 antagonistneurokinin₂ (±) 1-1,000 10-500   200 antagonist SR 48968 purine_(2X)PPADS 100-100,000 10,000- 50,000  antagonist 100,000 ATP- (−) pinacidil 1-10,000  100-1,000   500 sensitive K⁺ channel agonist Ca²⁺ channelnifedipine  1-10,000  100-5,000 1,000 antagonist kallikrein aprotinin0.1-1,000   50-500   200 inhibitor

E. Example V Alternate Irrigation Solution for Cardiovascular andGeneral Vascular Therapeutic and Diagnostic Procedures

The following drugs and concentration ranges in solution in aphysiologic carrier fluid are preferred for use in irrigating operativesites during cardiovascular and general vascular procedures. Again, thissolution is preferred relative to the solution set forth in Example IIabove for higher efficacy.

Concentration Class (Nanomolar): Most of Agent Drug TherapeuticPreferred Preferred serotonin₂ trazodone  0.1-2,000 50-500  200antagonist cyclo- ketorolac  100-10,000 500-5,000 3,000   oxygenaseinhibitor endothelin BQ 123 0.01-1,000   10-1,000 500 antagonist ATP-(−) pinacidil   1-10,000 100-1,000 500 sensitive K⁺ channel agonist Ca²⁺channel nisoldipine   1-10,000 100-1,000 500 antagonist nitric oxideSIN-1   10-10,000 100-1,000 500 donor

F. Example VI Alternate Irrigation Solution for Urologic Procedures

The following drugs and concentration ranges in solution in aphysiologic carrier fluid are preferred for use in irrigating operativesites during urologic procedures. The solution is believed to have evenhigher efficacy than the solution set forth in prior Example III.

Concentration Class (Nanomolar): Most of Agent Drug TherapeuticPreferred Preferred serotonin₂ LY 53857 0.1-500    1-100  50 antagonisthistamine₁ terfenadine 0.1-1,000   50-500 200 antagonist cyclooxy-ketorolac 100-100,000  500-5,000 3,000   genase inhibitor neurokinin₂ SR48968 1-1,000 10-500 200 antagonist purine_(2X) PPADS 100-100,00010,000- 50,000   antagonist 100,000 ATP- (−) pinacidil  1-10,000 100-1,000 500 sensitive K⁺ channel agonist nifedipine  1-10,000 100-5,000 1,000   Ca²⁺ channel antagonist kallikrein aprotinin0.1-1,000   50-500 200 inhibitor nitric oxide SIN-1 10-10,000  100-1,000500 donor

G. Example VII Cardiovascular and General Vascular Anti-RestenosisIrrigation Solution

The following drugs and concentration ranges in solution in aphysiologic carrier fluid are preferred for use in irrigation duringcardiovascular and general vascular therapeutic and diagnosticprocedures. The drugs in this preferred solution may also be added atthe same concentration to the cardiovascular and general vascularirrigation solutions of Examples II and V described above or ExampleVIII described below for preferred anti-spasmodic, anti-restenosis,anti-pain/anti-inflammation solutions.

Concentration (Nanomolar): Most Class of Agent Drug TherapeuticPreferred Preferred thrombin inhibitor hirulog  0.2-20,000  2-2,000 200glycoprotein IIb/IIIa integrelin  0.1-10,000 × Kd  1-1000 × Kd 100 × Kdreceptor blocker PKC inhibitor GF 109203X*  0.1-10,000  1-1,000 200protein tyrosine tyrphostin 10-100,000 100-20,000 10,000   kinaseinhibitor AG1296 *Also known as Go 6850 or Bisindoylmaleimide I(available from Warner-Lambert)

H. Example VIII Alternate Irrigation Solution for Cardiovascular andGeneral Vascular Therapeutic and Diagnostic Procedures

An additional preferred solution for use in cardiovascular and generalvascular therapeutic and diagnostic procedures is formulated the same asthe previously described formulation of Example V, except that thenitric oxide (NO donor) SIN-1 is replaced by a combination of twoagents, FK 409 (NOR-3) and FR 144420 (NOR-4), at the concentrations setforth below:

Concentration (Nanomolar): Most Class of Agent Drug TherapeuticPreferred Preferred NO donor FK 409  1-1,000  10-500   250 (NOR-3) NOdonor FR 144420 10-10,000 100-5,000 1,000 (NOR-4)

I. Example IX Alternate Irrigation Solution for Arthroscopy, GeneralSurgical Wounds, Burns and Oral/Dental Applications

An alternate preferred solution for use in irrigation of arthroscopic,general surgical and oral/dental applications is formulated the same asin the previously described Example IV, with the following substitution,deletion and additions at the concentrations set forth below:

1) amitriptyline is replaced by mepyramine as the H₁ antagonist;

2) the kallikrein inhibitor, aprotinin, is deleted;

3) a bradykinin₁ antagonist, [leu⁹] [des-Arg¹⁰] kalliden, is added;

4) a bradykinin₂ antagonist, HOE 140, is added; and

5) a μ-opioid agonist, fentanyl, is added.

Concentration (Nanomolar): Most Class of Agent Drug TherapeuticPreferred Preferred H₁antagonist mepyramine 0.1-1,000  5-200 100bradykinin₁ [leu⁹][des-Arg¹⁰] 0.1-500 10-200 100 antagonist kallidenbradykinin₂ HOE 140 1-1,000 50-500 200 antagonist μ-opioid fentanyl0.1-500 10-200 100 agonist

J. Example X Alternate Irrigation Solution for Urologic Procedures

An alternate preferred solution for use in irrigation during urologicprocedures is formulated the same as in the previously described ExampleVI with the following substitution, deletion and additions at theconcentrations set forth below:

1) SIN-1 is replaced as the NO donor by a combination of two agents: a)FK 409 (NOR-3); and b) FR 144420 (NOR-4);

2) the kallikrein inhibitor, aprotinin, is deleted;

3) a bradykinin₁ antagonist, [leu⁹] [des-Arg¹⁰] kalliden, is added; and

4) a bradykinin₂ antagonist, HOE 140, is added.

Concentration (Nanomolar): Most Class of Agent Drug TherapeuticPreferred Preferred NO donor FK 409 (NOR-3)  1-1,000  10-500 250 NOdonor FR 144420 10-10,000 100-5,000 1,000   (NOR-4) bradykinin₁[leu⁹]]des-Arg¹⁰]  0.1-500  10-200 100 antagonist kalliden bradykinin₂HOE 140  1-1,000  50-500 200 antagonist

K. Example XI Balloon Dilatation of Normal Iliac Arteries in the NewZealand White Rabbit and the Influence of Histamine/Serotonin ReceptorBlockade on the Response

The purpose of this study was twofold. First, a new in vivo model forthe study of arterial tone was employed. The time course of arterialdimension changes before and after balloon angioplasty is describedbelow. Second, the role of histamine and serotonin together in thecontrol of arterial tone in this setting was then studied by theselective infusion of histamine and serotonin receptor blocking agentsinto arteries before and after the angioplasty injury.

1. Design Considerations

This study was intended to describe the time course of change inarterial lumen dimensions in one group of arteries and to evaluate theeffect of histamine/serotonin receptor blockade on these changes in asecond group of similar arteries. To facilitate the comparison of thetwo different groups, both groups were treated in an identical mannerwith the exception of the contents of an infusion performed during theexperiment. In control animals (arteries), the infusion was normalsaline (the vehicle for test solution). The histamine/serotonin receptorblockade treated arteries received saline containing the receptorantagonists at the same rate and at the same part of the protocol ascontrol animals. Specifically, the test solution included: (a) theserotonin₃ antagonist metoclopramide at a concentration of 16.0 μM; (b)the serotonin₂ antagonist trazodone at a concentration of 1.6 μM; and(c) the histamine antagonist promethazine at concentrations of 1.0 μM,all in normal saline. Drug concentrations within the test solution were16-fold greater than the drug concentrations delivered at the operativesite due to a 16 to 1 flow rate ratio between the iliac artery (80 ccper minute) and the solution delivery catheter (5 cc per minute). Thisstudy was performed in a prospective, randomized and blinded manner.Assignment to the specific groups was random and investigators wereblinded to infusion solution contents (saline alone or saline containingthe histamine/serotonin receptor antagonists) until the completion ofthe angiographic analysis.

2. Animal Protocol

This protocol was approved by the Seattle Veteran Affairs Medical CenterCommittee on Animal Use and the facility is fully accredited by theAmerican Association for Accreditation of Laboratory Animal Care. Theiliac arteries of 3-4 kg male New Zealand white rabbits fed a regularrabbit chow were studied. The animals were sedated using intravenousxylazine (5 mg/kg) and ketamine (35 mg/kg) dosed to effect and a cutdownwas performed in the ventral midline of the neck to isolate a carotidartery. The artery was ligated distally, an arteriotomy performed and a5 French sheath was introduced into the descending aorta. Baseline bloodpressure and heart rate were recorded and then an angiogram of thedistal aorta and bilateral iliac arteries was recorded on 35 mm cinefilm (frame rate 15 per second) using hand injection of iopamidol 76%(Squibb Diagnostics, Princeton, N.J.) into the descending aorta. Foreach angiogram, a calibration object was placed in the radiographicfield of view to allow for correction for magnification when diametermeasurements were made. A 2.5 French infusion catheter (AdvancedCardiovascular Systems, Santa Clara, Calif.) was placed through thecarotid sheath and positioned 1-2 cm above the aortic bifurcation.Infusion of the test solution—either saline alone or saline containingthe histamine/serotonin receptor antagonists—was started at a rate of 5cc per minute and continued for 15 minutes. At 5 minutes into theinfusion, a second angiogram was performed using the previouslydescribed technique then a 2.5 mm balloon angioplasty catheter (theLightning, Cordis Corp., Miami, Fla.) was rapidly advanced underfluoroscopic guidance into the left and then the right iliac arteries.In each iliac the balloon catheter was carefully positioned between theproximal and distal deep femoral branches using bony landmarks and theballoon was inflated for 30 seconds to 12 ATM of pressure. The ballooncatheter was inflated using a dilute solution of the radiographiccontrast agent so that the inflated balloon diameter could be recordedon cine film. The angioplasty catheter was rapidly removed and anotherangiogram was recorded on cine film at a mean of 8 minutes after theinfusion was begun. The infusion was continued until the 15 minute timepoint and another angiogram (the fourth) was performed. Then theinfusion was stopped (a total of 75 cc of solution had been infused) andthe infusion catheter was removed. At the 30 minute time point (15minutes after the infusion was stopped), a final angiogram was recordedas before. Blood pressure and heart rate were recorded at the 15 and 30minute time points immediately before the angiograms. After the finalangiogram, the animal was euthanized with an overdose of the anestheticagents administered intravenously and the iliac arteries were retrievedand immersion fixed in formation for histologic analysis.

3. Angiographic Analysis

The angiograms were recorded on 35 mm cine film at a frame rate of 15per second. For analysis, the angiograms were projected from a Vanguardprojector at a distance of 5.5 feet. Iliac artery diameters atprespecified locations relative to the balloon angioplasty site wererecorded based on hand held caliper measurement after correction formagnification by measurement of the calibration object. Measurementswere made at baseline (before test solution infusion was begun), 5minutes into the infusion, immediately post balloon angioplasty (a meanof 8 minutes after the test solution was begun), at 15 minutes (justbefore the infusion was stopped) and at 30 minutes (15 minutes after theinfusion was stopped). Diameter measurements were made at three sites ineach iliac artery: proximal to the site of balloon dilatation, at thesite of balloon dilatation and just distal to the site of balloondilatation.

The diameter measurements were then converted to area measurements bythe formula:

Area=(Pi)(Diameter²)/4.

For calculation of vasoconstriction, baseline values were used torepresent the maximum area of the artery and percent vasoconstrictionwas calculated as: % Vasoconstriction={(Baseline area−Later time pointarea)/Baseline area}×100.

4. Statistical Methods

All values are expressed as mean ±1 standard error of the mean. The timecourse of vasomotor response in control arteries was assessed using oneway analysis of variance with correction for repeated measures. Post hoccomparison of data between specific time points was performed using theScheffe test. Once the time points at which significant vasoconstrictionoccurred had been determined in control arteries, the control andhistamine/serotonin receptor antagonist treated arteries were comparedat those time points where significant vasoconstriction occurred incontrol arteries using multiple analysis of variance with treatmentgroup identified as an independent variable. To compensate for theabsence of a single a priori stated hypothesis, a p value<0.01 wasconsidered significant. Statistics were performed using Statistica forWindows, version 4.5, (Statsoft, Tulsa, Okla.).

5. Results

The time course of arterial dimension changes before and after balloonangioplasty in normal arteries receiving saline infusion was evaluatedin 16 arteries from 8 animals (Table 23). Three segments of each arterywere studied: the proximal segment immediately upstream from the balloondilated segment, the balloon dilated segment and the distal segmentimmediately downstream from the balloon dilated segment. The proximaland distal segments demonstrated similar patterns of change in arterialdimensions: in each, there was significant change in arterial diameterwhen all time points were compared (proximal segment, p=0.0002 anddistal segment, p<0.001, ANOVA). Post hoc testing indicated that thediameters at the immediate post angioplasty time point weresignificantly less than the diameters at baseline or at the 30 minutetime point in each of these segments. On the other hand, the arterialdiameters in each segment at the 5 minute, 15 minute and 30 minute timepoints were similar to the baseline diameters. The balloon dilatedsegment showed lesser changes in arterial dimension than the proximaland distal segments. The baseline diameter of this segment was 1.82±0.05mm; the nominal inflated diameter of the balloon used for angioplastywas 2.5 mm and the actual measured inflated diameter of the balloon was2.20±0.03 mm (p<0.0001 vs. baseline diameter of the balloon treatedsegment). Thus, the inflated balloon caused circumferential stretch ofthe balloon dilated segment, but there was only slight increase in lumendiameter from baseline to the 30 minute time point (1.82±0.05 mm to1.94±0.07 mm, p=NS by post hoc testing).

TABLE 23 Angiographically determined lumen diameters at the specifiedtimes before and after balloon dilatation of normal iliac arteriesImmediate Segment Baseline 5 Minute Post PTA 15 Minute 30 MinuteProximal¹ 2.18 ± 0.7 2.03 ± 0.7 1.81 ± 0.08* 2.00 ± .08 2.23 ± .08Balloon² 1.82 ± .05 1.77 ± .03 1.79 ± .05 1.70 ± .04 1.94 ± .07 Distal³1.76 ± .04 1.68 ± .04** 1.43 ± .04* 1.54 ± .03 1.69 ± .06 Allmeasurements in mm. Means ± SEM. PTA = percutaneous transluminalangioplasty. ¹p = 0.0002 (ANOVA within group comparison), ²p = 0.03(ANOVA within group comparison), ³p < 0.0001 (ANOVA within groupcomparison). N = 16 at all time points. *p < 0.01 versus baseline and 30minute diameter measurements (Scheffe test for post hoc comparisons).**p < 0.01 verus immediate post PTA measurements (Scheffe test for posthoc comparisons). All other post hoc comparisons were not significantusing the p < 0.01 threshold.

Arterial lumen diameters were used to calculate lumen area then the areameasurements were used to calculate percent vasoconstriction bycomparison of the 5 minute, immediate post angioplasty, 15 and 30 minutedata to the baseline measurements. The proximal and distal segment dataexpressed as percent vasoconstriction are shown in FIG. 9; the changesin the amount of vasoconstriction over time are significant (in theproximal segment, p=0.0008; in the distal segment, p=0.0001, ANOVA).Post hoc testing identifies the vasoconstriction at the immediate postangioplasty time point as significantly different from that present atthe 30 minute time point (P<0.001 in both segments). In the distalsegment, the immediate post angioplasty vasoconstriction was alsosignificantly less than that at 5 minutes (p<0.01); no other differencesin intra-time point comparisons were significant by post hoc testing.

The luminal changes in control arteries can be summarized as follows: 1)Vasoconstriction with loss of approximately 30% of baseline luminal areaoccurs in the segments of artery proximal and distal to the balloondilated segment immediately after balloon dilatation. There are trendsto smaller amounts of vasoconstriction in the proximal and distalsegments before dilatation and at the 15 minute time point(approximately 7 minutes after dilatation) also but, by the 30 minutetime point (approximately 22 minutes after dilatation), a trend towardsvasodilatation has replaced the previous vasoconstriction; 2) In theballoon dilated segment, only minor changes in lumen dimensions arepresent, and, despite the use of a balloon with a significantly largerinflated diameter than was present in this segment at baseline, therewas no significant increase in lumen diameter of the dilated segment.These findings lead to a conclusion that any effects of the putativehistamine/serotonin treatment would only be detectable in the proximaland distal segments at the time points where vasoconstriction waspresent.

The histamine/serotonin receptor blockade solution was infused into 16arteries (8 animals); angiographic data was available at all time pointsin 12 arteries. Heart rate and systolic blood pressure measurements wereavailable in a subset of animals (Table 24). There were no differencesin heart rate or systolic blood pressure when the two animal groups werecompared within specific time points. Histamine/serotonin treatedanimals showed trends toward a decrease in the systolic blood pressurefrom baseline to 30 minutes (−14±5 mm Hg, p=0.04) and a lower heart rate(−26±10, p=0.05). Within the control animals, there was no change inheart rate or systolic blood pressure over the duration of theexperiment.

TABLE 24 Systolic blood pressure and heart rate measurements in controland histamine/serotonin treated animals Baseline 5 Minute 15 Minute 30Minute Group (N) (N) (N) (N) Systolic Blood Pressure Control  83 ± 4 (8) 84 ± 4 (8)  82 ± 6 (8)  80 ± 4 (8) Histamine/Serotonin  93 ± 5 (6)  87± 9 (4)  82 ± 9 (6)  80 ± 8 (6)* Heart Rate Control 221 ± 18 (5) 234 ±18 (4) 217 ± 23 (5) 227 ± 22 (5) Histamine/Serotonin 232 ± 8 (5) 232 ± 8(5) 209 ± 14 (5) 206 ± 12 (5)** Systolic blood pressure in mm Hg andheart rate in beats per minute. Mean ± SEM. *p = 0.04 for decrease insystolic blood pressure from baseline to 30 minutes and **p = 0.05 fordecrease in heart rate from baseline to 30 minutes within thehistamine/serotonin treated animals.

The proximal and distal segments of histamine/serotonin treated arterieswere compared to control arteries using the percent vasoconstrictionmeasurement. FIG. 10A shows the effects of the histamine/serotonininfusion on proximal segment vasoconstriction relative to thevasoconstriction present in the control arteries. When the findings inthe two treatment groups were compared at the baseline, immediate postangioplasty and 15 minute time points, histamine/serotonin infusionresulted in significantly less vasoconstriction compared to the controlsaline infusion (p=0.003. 2-way ANOVA). Comparison of the two treatmentgroups in the distal segment is illustrated in FIG. 10B. Despiteobserved differences in mean diameter measurements in the distalsegment, solution treated vessels exhibited less vasoconstriction thansaline treated control vessels at baseline, immediate post-angioplastyand 15 minute time points, this pattern did not achieve statisticalsignificance (p=0.32, 2-way ANOVA). Lack of statistical significance maybe attributed to smaller than expected vasoconstriction values in thecontrol vessels.

L. Example XII Amitriptyline Inhibition of 5-Hydroxytryptamine-InducedKnee Joint Plasma Extravasation-Comparison of Intra-Articular VersusIntravenous Routes of Administration

The following study was undertaken in order to compare two routes ofadministration of the 5-HT₂ receptor antagonist, amitriptyline: 1)continuous intra-articular infusion; versus 2) intravenous injection, ina rat knee synovial model of inflammation. The ability of amitriptylineto inhibit 5-HT-induced joint plasma extravasation by comparing both theefficacy and total drug dose of amitriptyline delivered via each routewas determined.

1. Animals

Approval from the Institutional Animal Care Committee at the Universityof California, San Francisco was obtained for these studies. MaleSprague-Dawley rats (Bantin and Kingman, Fremont, Calif.) weighing300-450 g were used in these studies. Rats were housed under controlledlighting conditions (lights on 6 A.M. to 6 P.M.), with food and wateravailable ad libitum.

2. Plasma Extravasation

Rats were anesthetized with sodium pentobarbital (65 mg/kg) and thengiven a tail vein injection of Evans Blue dye (50 mg/kg in a volume of2.5 ml/kg), which is used as a marker for plasma protein extravasation.The knee joint capsule was exposed by excising the overlying skin, and a30-gauge needle was inserted into the joint and used for the infusion offluid. The infusion rate (250 μl/min) was controlled by a SageInstruments Syringe pump (Model 341B, Orion Research Inc., Boston,Mass.). A 25-gauge needle was also inserted into the joint space andperfusate fluid was extracted at 250 μl/min, controlled by a SageInstruments Syringe pump (Model 351).

The rats were randomly assigned to three groups: 1) those receiving onlyintra-articular (IA) 5-HT (1 μM), 2) those receiving amitriptylineintravenously (IV) (doses ranging from 0.01 to 1.0 mg/kg) followed by IA5-HT (1 mM), and 3) those receiving amitriptyline intra-articularly (1A)(concentrations ranging from 1 to 100 nM) followed by IA 5-HT (1 μM)plus IA amitriptyline. In all groups, baseline plasma extravasationlevels were obtained at the beginning of each experiment by perfusing0.9% saline intra-articularly and collecting three perfusate samplesover a 15 min period (one every 5 min). The first group was thenadministered 5-HT IA for a total of 25 min. Perfusate samples werecollected every 5 min for a total of 25 min. Samples were then analyzedfor Evans Blue dye concentration by spectrophotometric measurement ofabsorbance at 620 nm, which is linearly related to its concentration(Carr and Wilhelm, 1964). The IV amitriptyline group was administeredthe drug during the tail vein injection of the Evans Blue dye. The kneejoints were then perfused for 15 min with saline (baseline), followed by25 min perfusion with 5-HT (1 μM). Perfusate samples were collectedevery 5 min for a total of 25 min. Samples were then analyzed usingspectrophotometry. In the IA amitriptyline group, amitriptyline wasperfused intra-articularly for 10 min after the 15 min saline perfusion,then amitriptyline was perfused in combination with 5-HT for anadditional 25 min. Perfusate samples were collected every 5 min andanalyzed as above.

Some rat knees were excluded from the study due to physical damage ofknee joint or inflow and outflow mismatch (detectable by presence ofblood in perfusate and high baseline plasma extravasation levels or kneejoint swelling due to improper needle placement).

a. 5-HT-Induced Plasma Extravasation

Baseline plasma extravasation was measured in all knee joints tested(total n=22). Baseline plasma extravasation levels were low, averaging0.022±0.003 absorbance units at 620 nm (average±standard error of themean). This baseline extravasation level is shown in FIGS. 11 and 12 asa dashed line.

5-HT (1 μM) perfused into the rat knee joint produces a time-dependentincrease in plasma extravasation above baseline levels. During the 25min perfusion of 5-HT intra-articularly, maximum levels of plasmaextravasation were achieved by 15 min and continued until the perfusionwas terminated at 25 min (data not shown). Therefore, 5-HT-inducedplasma extravasation levels reported are the average of the 15, 20 and25 min time points during each experiment. 5-HT-induced plasmaextravasation averaged 0.192±0.011, approximately an 8-fold stimulationabove baseline. This data is graphed in FIGS. 11 and 12, correspondingto the “0” dose of IV amitriptyline and the “0” concentration of IAamitriptyline, respectively.

b. Effect of Intravenous Amitriptyline on 5-HT-Induced PlasmaExtravasation

Amitriptyline administered via tail vein injection produced adose-dependent decrease in 5-HT-induced plasma extravasation as shown inFIG. 11. The IC₅₀ for IV amitriptyline inhibition of 5-HT-induced plasmaextravasation is approximately 0.025 mg/kg. 5-HT-induced plasmaextravasation is completely inhibited by an IV amitriptyline dose of 1mg/kg, the plasma extravasation averaging 0.034±0.010.

c. Effect of Intra-articular amitriptyline on 5-HT-Induced PlasmaExtravasation

Amitriptyline administered alone in increasing concentrationsintra-articularly did not affect plasma extravasation levels relative tobaseline, with the plasma extravasation averaging 0.018±0.002 (data notshown). Amitriptyline co-perfused in increasing concentrations with 5-HTproduced a concentration-dependent decrease in 5-HT-induced plasmaextravasation as shown in FIG. 12. 5-HT-induced plasma extravasation inthe presence of 3 nM IA amitriptyline was not significantly differentfrom that produced by 5-HT alone, however, 30 nM amitriptylineco-perfused with 5-HT produced a greater than 50% inhibition, while 100nM amitriptyline produced complete inhibition of 5-HT-induced plasmaextravasation. The IC₅₀ for IA amitriptyline inhibition of 5-HT-inducedplasma extravasation is approximately 20 nM.

The major finding of the present study is that 5-HT (1 μM) perfusedintra-articularly in the rat knee joint produces a stimulation of plasmaextravasation that is approximately 8-fold above baseline levels andthat either intravenous or intra-articular administration of the 5-HT₂receptor antagonist, amitriptyline, can inhibit 5-HT-induced plasmaextravation. The total dosage of administered amitriptyline, however,differs dramatically between the two methods of drug delivery. The IC₅₀for IV amitriptyline inhibition of 5-HT-induced plasma extravasation is0.025 mg/kg, or 7.5×10⁻³ mg in a 300 g adult rat. The IC₅₀ for IAamitriptyline inhibition of 5-HT-induced plasma extravasation isapproximately 20 nM. Since 1 ml of this solution was delivered everyfive minutes for a total of 35 min during the experiment, the totaldosage perfused into the knee was 7 ml, for a total dosage of 4.4×10⁻⁵mg perfused into the knee. This IA amitriptyline dose is approximately200-fold less than the IV amitriptyline dose. Furthermore, it is likelythat only a small fraction of the IA perfused drug is systemicallyabsorbed, resulting in an even greater difference in the total delivereddose of drug.

Since 5-HT may play an important role in surgical pain and inflammation,as discussed earlier, 5-HT antagonists such as amitriptyline may bebeneficial if used during the perioperative period. A recent studyattempted to determine the effects of oral amitriptyline onpost-operative orthopedic pain (Kerrick et al., 1993). An oral dose aslow as 50 mg produced undesirable central nervous system side-effects,such as a “decreased feeling of well-being”. Their study, in addition,also showed that oral amitriptyline produced higher pain scale scoresthan placebo (P<0.05) in the post-operative patients. Whether this wasdue to the overall unpleasantness produced by oral amitriptyline is notknown. In contrast, an intra-articular route of administration allows anextremely low concentration of drug to be delivered locally to the siteof inflammation, possibly resulting in maximal benefit with minimalside-effects.

M. Example XIII Effects Of Cardiovascular and General Vascular SolutionOn Rotational Atherectomy-Induced Vasospasm In Rabbit Arteries

1. Solution Tested

This study utilized an irrigation solution consisting of the agents setforth in Example V. above, with the following exceptions. Nitroprussidereplaced SIN-1 as the nitric oxide donor and nicardipine replacednisoldipine as the Ca²⁺ channel antagonist.

The concentration of nitroprusside was selected based on itspreviously-defined pharmacological activity (EC₅₀). The concentrationsof the other agents in this test solution were determined based on thebinding constants of the agents with their cognate receptors.Furthermore, all concentrations were adjusted based on a blood flow rateof 80 cc per minute in the distal aorta of the rabbit and a flow rate of5 cc per minute in the solution delivery catheter. Three components weremixed in one cc or less DMSO, and then these components and theremaining three components were mixed to their final concentrations innormal saline. A control solution consisting of normal saline wasutilized. The test solution or the control solution was infused at arate of 5 cc per minute for 20 minutes. A brief pause in the infusionwas necessary at the times blood pressure measurements were made, soeach animal received about 95 cc of the solution in the 20 minutetreatment period.

2. Animal Protocol

This protocol was approved by the Seattle Veteran Affairs Medical CenterCommittee on Animal Use, which is accredited by the American Associationfor Accreditation of Laboratory Animal Care. The iliac arteries of 3-4kg male New Zealand white rabbits fed a 2% cholesterol rabbit chow for3-4 weeks were studied. The animals were sedated using intravenousxylazine (5 mg/kg) and ketamine (35 mg/kg) dosed to effect and a cutdownwas performed in the ventral midline of the neck to isolate a carotidartery. The artery was ligated distally, an arteriotomy performed and a5 French sheath was introduced into the descending aorta and positionedat the level of the renal arteries. Baseline blood pressure and heartrate were recorded. An angiogram of the distal aorta and bilateral iliacarteries was recorded on 35 mm cine film (frame rate 15 per second)using hand injection of iopamidol 76% (Squibb Diagnostics, Princeton,N.J.) into the descending aorta.

For each angiogram, a calibration object was placed in the radiographicfield of view to allow for correction for magnification when diametermeasurements were made. Infusion of either the above described testsolution or a saline control solution was started through the side armof the 5 French sheath (and delivered to the distal aorta) at a rate of5 cc per minute and continued for 20 minutes. At 5 minutes into theinfusion, a second angiogram was performed using the previouslydescribed technique. Then a 1.25 mm or a 1.50 mm rotational atherectomyburr (Heart Technology/Boston Scientific Inc.) was advanced to the iliacarteries. The rotational atherectomy burr was advanced three times overa guide wire in each of the iliac arteries at a rotation rate of 150,000to 200,000 RPM. In each iliac, the rotational atherectomy burr wasadvanced from the distal aorta to the mid portion of the iliac arterybetween the first and second deep femoral branches. The rotationalatherectomy burr was rapidly removed and another angiogram was recordedon cine film at a mean of 8 minutes after the infusion was begun.

The infusion was continued until the 20 minute time point, and anotherangiogram (the fourth) was performed. Then the infusion was stopped. Atotal of about 95 cc of the control or test solution had been infused.At the 30 minute time point (15 minutes after the infusion was stopped),a final angiogram was recorded as before. Blood pressure and heart ratewere recorded at the 15 and 30 minute time points immediately before theangiograms. After the final angiogram, the animal was euthanized with anoverdose of the anesthetic agents administered intravenously.

3. Angiographic Analysis

The angiograms were recorded on 35 mm cine film at a frame rate of 15per second. Angiograms were reviewed in random order without knowledgeof treatment assignment. For analysis, the angiograms were projectedfrom a Vanguard projector at a distance of 5.5 feet. The entireangiogram for each animal was reviewed to identify the anatomy of theiliac arteries and to identify the sites of greatest spasm in the iliacarteries. A map of the iliac anatomy was prepared to assist inconsistently identifying sites for measurement. Measurements were madeon the 15 minute post rotational atherectomy angiogram first, then inrandom order on the remaining angiograms from that animal. Measurementswere made using an electronic hand-held caliper (Brown & Sharpe, Inc.,N. Kingston, R.I.). Iliac artery diameters were measured at threelocations: proximal to the first deep femoral branch of the iliacartery; at the site of most severe spasm (this occurred between thefirst and second deep femoral artery branches in all cases); and at adistal site (near or distal to the origin of the second deep femoralartery branch of the iliac artery). Measurements were made at baseline(before test solution infusion was begun), 5 minutes into the infusion,immediately post rotational atherectomy (a mean of 8 minutes after thetest solution was begun), at 20 minutes just after the infusion wasstopped (this was minutes after the rotational atherectomy was begun)and at 15 minutes after the infusion was stopped (30 minutes after therotational atherectomy was begun). The calibration object was measuredin each angiogram.

The diameter measurements were then converted to area measurements bythe formula:

Area=(Pi)(Diameter²)/4.

For calculation of vasoconstriction, baseline values were used torepresent the maximum area of the artery and percent vasoconstrictionwas calculated as:

% Vasoconstriction={(Baseline area−Later time point area)/Baselinearea}×100.

4. Statistical Methods

All values are expressed as mean±1 standard error of the mean. The timecourse of vasomotor response in control arteries was assessed using oneway analysis of variance with correction for repeated measures. Post hoccomparison of data between specific time points was performed using theScheffe test. Test solution treated arteries were compared to salinetreated arteries at specified locations in the iliac arteries and atspecified time points using multiple analysis of variance (MANOVA). Tocompensate for the absence of a single a priori hypothesis, a pvalue<0.01 was considered significant. Statistics were performed usingStatistica for Windows, version 4.5, (Statsoft, Tulsa, Okla.).

5. Results

Eight arteries in 4 animals received saline solution and 13 arteries inseven animals received test solution. In each artery, regardless of thesolution used, rotational atherectomy was performed with the rotatingburr passing from the distal aorta to the mid-portion of the iliacartery. Thus, the proximal iliac artery segment and the segmentdesignated as the site of maximal vasoconstriction were subjected to therotating burr. The guide wire for the rotational atherectomy catheterpassed through the distal segment, but the rotating burr of therotational atherectomy catheter itself did not enter the distal segment.

Iliac artery diameters in saline treated arteries at the three specifiedsegments are summarized in Table 25. In the proximal segment, there wasno significant change in the diameter of the artery over the time courseof the experiment (p=0.88, ANOVA). In the mid-iliac artery at the siteof maximal vasoconstriction, there was a significant reduction indiameter with the largest reduction occurring at the 15 minutepost-rotational atherectomy time point (p<0.0001, ANOVA comparingmeasurements at all 5 time points). The distal segment diameter did notsignificantly change over the time course of the experiment (p=0.19,ANOVA comparing all time points) although there was a trend towards asmaller diameter at the immediate post- and 15 minute post-rotationalatherectomy time points.

TABLE 25 Iliac artery lumen diameters at specified time points in salinetreated arteries 5 Minutes Immediate 15 Minute 30 Minutes Baseline intoInfusion Post RA after RA after RA Segment N = 8 N = 8 N = 8 N = 8 N = 8Proximal¹ 2.40 ± .18 2.32 ± .14 2.32 ± 0.13 2.38 ± .13 2.34 ± .07* Mid²2.01 ± .08 1.84 ± .09 1.57 ± .15 1.24 ± .13 1.87 ± .06** Distal³ 2.01 ±.10 1.86 ± .08 1.79 ± .08 1.81 ± .09 1.96 ± .06*** RA = rotationalatherectomy ¹Proximal iliac artery measurement site, proximal to thefirst deep femoral branch ²Mid iliac artery at the site of maximalvasospasm ³Distal iliac artery measurement site, near or distal to thesecond deep femoral branch *p = 0.88 by ANOVA comparing diameters in theproximal segment at the five time points. ***p = 0.000007 by ANOVAcomparing diameters at site of maximal vasospasm at the five timepoints. ***p = 0.19 by ANOVA comparing diameters in the distal segmentat the five time points.

The diameters of iliac arteries treated with the test solution are shownin Table 26. Angiograms were not recorded in three of these arteries atthe 5 minute post-initiation of the infusion time point and angiographicdata were excluded from two arteries (one animal) at the 30 minutepost-rotational atherectomy time point because the animal received anair embolus at the 15 minute angiogram that resulted in hemodynamicinstability. Because there is a variable number of observations at thefive time points, no ANOVA statistic was applied to this data. Still itis apparent that the magnitude of change in the diameter measurementswithin segments in the test solution treated arteries over the timecourse of the experiment is less than was seen in the saline treatedarteries.

TABLE 26 Iliac artery lumen diameters at specified time points in TestSolution treated arteries 5 Minutes Immediate 15 Minute 30 MinutesBaseline into Infusion Post RA after RA after RA Segment N = 13 N = 10 N= 13 N = 13 N = 11 Proximal¹ 2.28 ± .06 2.07 ± .07 2.22 ± .05 2.42 ± .062.39 ± .08 Mid² 1.97 ± .06 1.79 ± .06 1.74 ± .04 1.95 ± .07 1.93 ± .08Distal³ 2.00 ± .06 1.92 ± .04 1.90 ± .04 2.00 ± .06 2.01 ± .07 RA =rotational atherectomy ¹Proximal iliac artery measurement site, proximalto the first deep femoral branch ²Mid iliac artery at the site ofmaximal vasospasm ³Distal iliac artery measurement site, near or distalto the second deep femoral branch

Because of the different number of observations at the various timepoints, ANOVA was not performed to determine the statisticalsimilarity/difference in diameters within specific segments.

The primary endpoint for this study was the comparison of the amounts ofvasoconstriction in saline treated and test solution treated arteries.Vasoconstriction was based on arterial lumen areas derived from arterydiameter measurements. Area values at the 5 minute, immediatepost-rotational atherectomy and later time points were compared to thebaseline area values to calculate the relative change in area. Theresults were termed “vasoconstriction” if the lumen area was smaller atthe later time point than at baseline, and “vasodilatation” if the lumenarea was larger at the later time point compared to the baseline area(Tables 27 and 28). To facilitate statistical analysis with the largestnumber of observations possible in both treatment groups, the testsolution and saline treated artery data were compared at the immediatepost- and at the 15 minute postrotational atherectomy time points.

In the proximal segment (FIG. 13), there was essentially no change inlumen area with either treatment at the immediate post-rotationalatherectomy time point, but there was some vasodilatation in thissegment by the 15 minute post-rotational atherectomy time point. Testsolution did not alter the results of rotational atherectomy compared tosaline treatment in this segment. In the mid-vessel (FIG. 14) at thesite of maximal vasoconstriction however, test solution significantlyblunted the vasoconstriction, caused by rotational atherectomy in thesaline treated arteries (p=0.0004, MANOVA corrected for repeatedmeasures). In the distal segment (FIG. 15), there was littlevasoconstriction in the saline treated arteries and test solution didnot significantly alter the response to rotational atherectomy.

TABLE 27 Amount of vasoconstriction (negative values) or vasodilatation(positive values) at specified time points in saline treated arteries. 5Minutes Immediate 15 Minute 30 Minutes into Infusion Post RA after RAafter RA Segment N = 8 N = 8 N = 8 N = 8 Proximal¹ −3% ± −1% ±  3% ±  3%± 13% .8% 10% 8% Mid² −14% ± −35% ± −58% ± −11% ± .9% 7% 10% 7% Distal³−9% ± −14% ± −14% ±  2% ± .12% .10% .14% 10% ¹Proximal iliac arterymeasurement site, proximal to the first deep femoral branch ²Mid iliacartery at the site of maximal vasospasm ³Distal iliac artery measurementsite, near or distal to the second deep femoral branch

TABLE 28 Amount of vasoconstriction (negative values) or vasodilatation(positive values) at specified time points in Test Solution treatedarteries. 5 Minutes Immediate 15 Minute 30 Minutes into Infusion Post RAafter RA after RA Segment N = 10 N = 13 N = 13 N = 11 Proximal¹ −17% ± −4% ±  14% ± 7% ± 9% .5% 3%   6% Mid² −14% ± −20% ± 0.3% ± −5% ± .5% 5%5%   7% Distal³  −8% ± −9% ±   1% ± 3% ± .6% .4% .4%   4% ¹Proximaliliac artery measurement site, proximal to the first deep femoral branch²Mid iliac artery at the site of maximal vasospasm ³Distal iliac arterymeasurement site, near or distal to the second deep femoral branch

The hemodynamic response in the saline and test solution treatedarteries is summarized in Table 29. Compared to saline treated animals,test solution treated animals sustained substantial hypotension andsignificant tachycardia during the solution infusion. By 15 minutesafter completion of the infusion (or at the 30 minute postrotationalatherectomy time point), test solution treated animals showed somepartial, but not complete, return of blood pressure towards baseline.

TABLE 29 Blood pressure and heart rates during the protocol. Base- 5 1530 line Minute Minute Minute Group (N) (N) (N) (N) Systolic BloodPressure Saline  83 ±  93 ±  92 ±  83 ± 10 (4)*  9 (4)  6 (3)  11 (4)Test Solution  92 ±  35 ±  35 ±  46 ± 5 (7)**  5 (7)  5 (7)  5 (7) HeartRate Saline 202 ± 204 ± 198 ±  19 ± 29 (3)*  16 (3)  3 (3)  22 (3) TestSolution 187 ± 246 ± 240 ± 247 ± 16 (7)** 111 (7)  11 (7)  5 (7) *Therewas no significant change in systolic blood pressure or heart rate inthis group (p = 0.37 for systolic blood pressure and p = 0.94 for heartrate, ANOVA). **There was a highly significant change in systolic bloodpressure and heart rate in this group (p < 0.0001 for systolic bloodpressure and p = 0.002 for heart rate, ANOVA).

6. Summary of Study

1. Rotational atherectomy in hypercholesterolemic New Zealand whiterabbits results in prominent vasospasm in the mid-portion of iliacarteries subjected to the rotating burr. The vasospasm is most apparent15 minutes after rotational atherectomy treatment and has almostcompletely resolved without pharmacologic intervention by 30 minutesafter rotational atherectomy.

2. Under the conditions of rotational atherectomy treatment studied inthis protocol, test solution treatment in accordance with the presentinvention almost completely abolishes the vasospasm seen after themid-iliac artery is subjected to the rotating burr.

3. Treatment with test solution of the present invention given theconcentration of components used in this protocol results in profoundhypotension during the infusion of the solution. The attenuation ofvasospasm after rotational atherectomy by test solution occurred in thepresence of severe hypotension.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes to the disclosedsolutions and methods can be made therein without departing from thespirit and scope of the invention. For example, alternate paininhibitors and anti-inflammation and anti-spasm and anti-restenosisagents may be discovered that may augment or replace the disclosedagents in accordance with the disclosure contained herein. It istherefor intended that the scope of letters patent granted hereon belimited only by the definitions of the appended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of preemptivelyinhibiting pain and inflammation at a wound during a surgical procedure,comprising delivery to a wound during a surgical procedure a dilutesolution comprising no more than 100,000 nanomolar concentration of anopioid receptor agonist in a liquid carrier, wherein the solution isapplied locally to the surgical site.
 2. The method of claim 1, whereinthe solution is applied perioperatively to the surgical site.
 3. Themethod of claim 2, wherein the solution is applied continuously to thesurgical site.
 4. The method of claim 1, wherein the solution comprises0.1-10,000 times K_(d) of the opioid receptor agonist.
 5. The method ofclaim 4, wherein the solution comprises 1.0-1,000 times K_(d) of theopioid receptor agonist.
 6. The method of claim 5, wherein the solutioncomprises approximately 100 times K_(d) of the opioid receptor agonist.7. The method of claim 1, wherein the solution is applied locally to thesurgical site during an arthroscopic procedure.
 8. The method of claim1, wherein the solution is applied locally to the surgical site during aurologic procedure.
 9. The method of claim 1, wherein the opioidreceptor agonist is selected from among μ-opioid receptor agonists,δ-opioid receptor agonists, and κ-opioid receptor agonists.
 10. Themethod of claim 9, wherein the opioid receptor agonist is an μ-opioidreceptor agonist.
 11. The method of claim 10, wherein the solutioncomprises 0.1-500 nanomolar of the μ-opioid receptor agonist.
 12. Themethod of claim 10, wherein the μ-opioid receptor agonist is PL
 017. 13.The method of claim 12, wherein the solution comprises 0.05-50 nanomolarPL
 017. 14. The method of claim 13, wherein the solution comprises0.25-10 nanomolar PL
 017. 15. The method of claim 10, wherein theμ-opioid receptor agonist is Tyr-D-Ala-Gly-[N-MePhe]-NH(CH₂)—OH.
 16. Themethod of claim 15, wherein the solution comprises 0.1-100 nanomolarTyr-D-Ala-Gly-[N-MePhe]-NH(CH₂)—OH.
 17. The method of claim 16, whereinthe solution comprises 0.5-20 nanomolarTyr-D-Ala-Gly-[N-MePhe]-NH(CHV)—OH.
 18. The method of claim 10, whereinthe μ-opioid receptor agonist is sufentanyl.
 19. The method of claim 18,wherein the solution comprises 0.01-50 nanomolar sufentanyl.
 20. Themethod of claim 19, wherein the solution comprises 1-20 nanomolarsufentanyl.
 21. The method of claim 10, wherein the μ-opioid receptoragonist is fentanyl.
 22. The method of claim 21, wherein the solutioncomprises 0.1-500 nanomolar fentanyl.
 23. The method of claim 22,wherein the solution comprises 10-200 nanomolar fentanyl.
 24. The methodof claim 23, wherein the solution comprises 100 nanomolar fentanyl. 25.The method of claim 9, wherein the opioid receptor agonist is anδ-opioid receptor agonist.
 26. The method of claim 25, wherein thesolution comprises 0.1-500 nanomolar of the δ-opioid receptor agonist.27. The method of claim 25, wherein the δ-opioid receptor agonist is[D-Pen²,D-Pen⁵]enkephalin.
 28. The method of claim 27, wherein thesolution comprises 0.1-500 nanomolar of [D-Pen²,D-Pen⁵]enkephalin. 29.The method of claim 28, wherein the solution comprises 1.0-100 nanomolarof [D-Pen²,D-Pen⁵]enkephalin.
 30. The method of claim 9, wherein theopioid receptor agonist is a κ-opioid receptor agonist.
 31. The methodof claim 30, wherein the solution comprises 0.1-500 nanomolar of theκ-opioid receptor agonist.
 32. The method of claim 30, wherein theκ-opioid receptor agonist is(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetaminde.
 33. The method of claim 32, wherein the solution comprises0.1-500 nanomolar of(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide.
 34. The method of claim 33, wherein the solution comprises1.0-100 nanomolar of(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide.